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This thesis describes novel research using Portobello mushroom spores (PMS) as a biotemplate and reducing agent that may be used to produce nanocomposites and replicas with societal benefits. First, the use of PMS as a green eco-friendly bioreagent for the biosynthesis of Ag, Au and TiOx nanoparticles (NPs) and their hybrids is described. These have been characterised using SEM, TEM, XRD, FTIR, UV, SIMS, DLS and TGA (where a full list of acronyms is to be found on page iv). The spherical shape, location and mean diameter of the PMS-held (5-30nm) Ag and Au NPs were confirmed. PMS functional groups are comparable with those in plant sources and allow them to act as reducing/capping/stabilising agents. Second, the sequential biosynthesis of PMS-held Ca2+, CaCO3 and nanohydroxyapatite (nHAp) is described where surface nucleation and growth are facilitated by biotemplate surface hydrophilic polar groups (-OH and -COO-). XRD and HRTEM suggest that the nHAp so formed has lower crystallinity and greater directed growth in the (002) direction than commercial nHAp (e.g. Bio-Oss), making PMS-routes useful for the delivery of artificial bone (after subsequent PMS removal). Furthermore, PMS-held nHAp showed more rapid in-vitro mineralization in simulated body fluid (SBF). Third, it is shown that ascorbic acid (AA), haemoglobin (Hb) and insulin (In) can be loaded into/released by PMS through its cell walls faster than with synthetic hydrogels (PVA); the transdermal use of PMS nanocomposites is considered in the context of drug delivery nanotechnology, avoiding size-dependent toxicity. Fourth, it is shown by traditional diffusion and novel methods that the antimicrobial activity especially of Ag/PMS, but also TiOx/PMS and Ag-TiOx/PMS (but not Au/PMS) is good in the inhibition of the growth of E.coli and S.aureus bacteria. The highest activity of Ag/PMS is attributed to Agx+ release. Fifth, the synthesis of NPs/PMS and biomimetic TiOx/PMS has shown to lead to good photocatalysis for the removal of coloured organic pollutants (e.g. methyl orange) from water, with faster rates of removal in molecules/mg/s and turnover numbers (TON) (s-1) than commercial anatase-rutile TiO2 (P25) possibly as the texture of the titania replicas raises the number of multiple reflections of light.

Esporos de Bacillus subtilis apresentam propriedades adjuvantes, sendo capazes de aumentar a resposta humoral após a sua coadministração com antígenos misturados ou adsorvidos à sua superfície. Mas, para isso, é necessária a produção de esporos altamente purificados e com rendimentos elevados. Neste trabalho, realizamos com sucesso uma análise quantitativa das condições de esporulação e dos métodos de purificação, o que melhorou a reprodutibilidade do processo e a obtenção de amostras com elevado grau de pureza e rendimento. Avaliamos também as propriedades imunomodulatórias destes esporos, utilizando como antígeno modelo a proteína recombinante Gag-p24 do HIV-1. A coadministração, mas não a adsorção à superfície do esporo, aumentou a imunogenicidade do antígeno sem induzir efeitos deletérios após a administração parenteral em camundongos BALB/c e C57BL/6. Além de promoveram a ativação das APCs, os esporos interagem com receptores relacionados à imunidade inata, devido à ausência do efeito adjuvante em camundongos nocautes para TLR2. Esses resultados abrem perspectivas interessantes para a utilização de esporos como adjuvantes vacinais. / Bacillus subtilis spores have been shown to behave as vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. Nonetheless, such specialized application requires highly purified spore preparations at high yields. In this work, we successfully performed a systematic quantitative analysis of sporulation conditions and spore purification methods, which improved the reproducibility of the process and the obtainment of samples with high purity and yield. Afterwards, we further evaluated the immune modulatory properties of these spores using a recombinant HIV-1 Gag-p24 protein as a model antigen. The co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen, without inducing deleterious effects, after subcutaneous administration to BALB/c and C57BL/6 mice. Besides promoting activation of antigen presenting cells, spores interact with receptors related to innate immunity, due to the absence of the adjuvant effect on TLR2 knockout mice. These results open interesting perspectives for the use of B. subtilis spores as vaccine adjuvants.

Em busca de melhorar a eficiência do uso de fontes de fósforo (P) e viabilizar o aumento da absorção deste nutriente, objetivou-se com este trabalho determinar as mudanças promovidas por plantas de cobertura, associadas à fontes fosfatadas de solubilidade distintas, na colonização por fungos micorrízicos arbusculares (FMA), como também avaliar as mudanças promovidas na disponibilidade de frações de P no solo e na eficiência de uso do P pelas coberturas. O delineamento adotado foi de blocos ao acaso, em esquema fatorial 5x3, com quatro repetições. Foram utilizadas quatro plantas de cobertura: milheto (Pennisetum glaucum), braquiária (Brachiaria ruziziensis), guandú-anão (Cajanus cajan) e crotalária (Crotalária juncea), além do controle (pousio);Duas fontes de P: fosfato natural reativo Bayóvar e superfosfato simples, além de um tratamento controle (sem fosfato),. Avaliou-se a colonização micorrízica por FMA e densidade de esporos no solo em três épocas, correspondendo à estação chuvosa e seca de 2013 e a estação chuvosa de 2014. Avaliou-se também a produção de massa seca e o acúmulo de P pelas plantas. No final do segundo ano, analisou-se o fracionamento de P do solo. A colonização radicular foi mais influenciada nas épocas chuvosas. A presença das plantas de cobertura proporcionou maior colonização micorrízica e densidade de esporos na época chuvosa. As aplicações de fontes fosfatadas não influenciam a densidade dos esporos, enquanto que as plantas de cobertura, principalmente o guandú e a braquiária, apresentaram aumento no número de esporos. Apesar de não aumentar as frações lábeis do P no solo, a utilização de plantas de cobertura proporcionou maior ciclagem do nutriente. Os fosfatos não influenciaram a massa seca nem o acúmulo de P, sendo que houve efeitos das plantas de cobertura na massa seca e no acúmulo de P na parte aérea, com destaque para o milheto e a braquiária. / In order to improve the use efficiency of phosphorus sources and enable a higher absorption of this nutrient, this research aimed to determine the changes promoted by cover crops and phosphorus sources of different solubility in the colonization by arbuscular mycorrhizal fungi (AMF), as much as the availability of P fractions in the soil and the use efficiency of P by the cover crops. A complete randomized block design was adopted, in a 5x3 factorial scheme, with four replications. For the first factor were used four cover crops: millet (Pennisetum glaucum), brachiaria (Brachiaria ruziziensis), pigeon pea (Cajanus cajan) and sun hemp (Crotalaria juncea), including a control (fallow); for the second factor were used the reactive phosphate Bayovar and the simple superphosphate plus a control treatment (no phosphate). It was assessed the AMF colonization, the spore density in the soil in three different times, the rainy and dry seasons in 2013, and the rainy season in 2014. At the end of 2014, it was also evaluated the amount of the fractioned P in the soil. The root colonization was more influenced in rainy season. The presence of cover crops provided greater mycorrhizal colonization and spore density in the rainy season. The applications of phosphate sources did not influence the spores density. However, the cover crops, mainly pigeon pea and brachiaria, showed an increase in the number of spores. The use of cover crops did not increase the labile fractions of P in the soil, Nevertheless they provided greater cycling of this nutrient. Phosphates did not influence the dry matter or the accumulation of P. Interestingly, the dry matter and P accumulation in shoots was influenced by the kind of cover crops, being higher for millet and brachiaria.

O leite produzido a ultra alta temperatura denominado oficiamente como UHT é o leite de maior aceitação no mercado consumidor brasileiro. Com a finalidade de ampliar o conhecimento sobre a segurança microbiológica do leite UHT, objetivou-se estudar tanto os Bacillus spp, que vêm sendo relatados como contaminantes do leite UHT no Brasil, como os eventuais esporos sobreviventes ao processamento. A partir de 65 análises de leite UHT para a determinação de microrganismos mesófilos aeróbios estritos e facultativos viáveis, no leite UHT procedente das indústrias sob Inspeção Federal no estado de São Paulo, 7 apresentaram contagens acima de 100 UFC/ml, indicando um percentual de 10,76 % de provas fora dos padrões para essa determinação, no período de jul a nov de 2003. Entretanto por atuação dos controles de qualidade das indústrias partidas foram inutilizadas e o leite que foi ao consumo apresentou 7,69% de microrganismos mesofílicos aeróbios acima dos padrões, sendo que 6.15% destes microrganismos foram considerados como Bacillus sporothermodurans. Dos isolamentos das contagens de microrganismos mesófilos acima dos padrões, foram efetuados estudos de seis cepas de Bacillus spp do leite UHT das indústrias, além de três cepas isoladas do leite UHT alvo de reclamações de consumidores procedente do Instituto Adolfo Lutz da capital de São Paulo. O objetivo do presente projeto foi, após a identificação cultural, microscópica, bioquímica e genética de culturas do Bacillus spp, estudar a patogenicidade das amostras em diversos modelos experimentais como: Teste do camundongo neonatos, alça ligada de coelho, inoculação intraperitoneal em camundongo BALB C e culturas celulares (Vero, HEp2 e Fibroblastos de Embrião de Galinha). As 3 amostras isoladas de leite de reclamações dos consumidores do Instituto Adolfo Lutz de São Paulo foram tipificadas preliminarmente pela técnica de Espectroscopia Infra-Vermelho a Transformada Fourier (FT- IR) como Bacillus flexus, enquanto que as amostras isoladas do leite procedente de três indústrias de São Paulo apresentaram presença de B. flexus e B.oleronius e Bacillus sporothermodurans. Exemplares de B.flexus testados quanto a patogenicidade apresentaram efeito citopático nas culturas celulares Vero, HEP2 e Fibroblastos de Embrião de Galinha e morbidade em camundongo BALB C. O Bacillus sporothermodurans não apresentou patogenicidade a nenhum dos modelos biológicos citados. / The milk produced at ultra high temperature, denominated UHT, is the most popular milk in the Brazilian market. With the intention of increasing the knowledge of microbiologic safety in UHT milk, we concentrated our study on the Bacillus spp, described as the most important contaminants of UHT milk in Brazil, and on possible survivors of the processing procedures. From the 65 analysis of UHT milk collected to determine the presence of strict, aerobe, mesophile microorganisms and viable options, coming from industries under Federal Inspection in São Paulo state, 7 presented a microbial count higher than 100CFU/ml, indicating a percentage of 10.76% samples outside the standard for this determination, in the period between July and November 2003. However, due to the quality control in industries, batches were rendered useless and the milk that went for consumption presented 7.69% mesophile microorganisms above standard, being these organisms 6.15% was considered Bacillus sporothermodurans. From the isolation of mesophile Bacillus counts above average, 5 strains Bacillus spp species from UHT industries were studied, as well as 3 strains isolated from UHT milk denounced by consumers of São Paulo city\'s Adolfo Lutz Institute. The aim of the project, following cultural, microscopic, biochemical and genetic identification of the Bacillus spp, was to study pathogenicity in samples from several experimental models, such as: neonatal mice; rabbit ileum loops, intraperitoneal inoculation of BALB C mice and cell cultures: Vero, Hep2 and Chick Embryo Fibroblasts. The three isolated samples of milk denounced by São Paulo consumers were typified as Bacillus flexus by the Fourier Infrared Transformed Espectroscopy (IF-FR) technique, while isolated samples of milk from 3 industries in São Paulo, accused the presence of the B. flexus, B. oleronius and Bacillus sporothermodurans. Samples of the B. flexus tested in terms of pathogenicity presented a citopatic effect upon cell cultures Vero, HeP2 and Chick Embryo Fibroblasts and a morbidity in mice BalbC. The Bacillus Sporothermodurans did not present pathogenicity in any of the biological models mentioned.

The causative agent of the most devastating honeybee disease, American foulbrood (AFB), is the spore-forming bacterium Paenibacillus larvae. To prevent AFB outbreaks beekeepers prophylactically treat their hives with antibiotics even though it decreases the overall health of uninfected hives. A new treatment for AFB is needed due to recent legislation against using antibiotics, antibiotic resistance developing in P. larvae, and the resilience of P. larvae spores. Bacteriophages, or phages, are an attractive alternative to traditional antibiotics because of their specificity and ability to evolve alongside their target bacterium. In this study, two phage cocktails were developed for the treatment of AFB. The first cocktail was comprised of Brevibacillus laterosporus phages. B. laterosporus is a commensal microbe in most honeybee guts. When treated with B. laterosporus phages, B. laterosporus is induced to produce an antimicrobial toxin to which P. larvae is highly sensitive. Treating AFB infected hives with B. laterosporus phages was able to clear active infections at a rate of 75% as opposed to untreated hives that did not recover. However, B. laterosporus phages did not clear latent P. larvae spores and recovered hives relapsed after treatment. The second cocktail was comprised of P. larvae phages and hives treated with the second cocktail recovered at a rate of 100%, protected 100% of at-risk hives, and treated hives did not relapse with AFB suggesting neutralization of P. larvae spores. A P. larvae phage used in the second cocktail was examined to identify any spore-phage interactions. Results from modified plaque assays, fluorescence from FITC-labeled phages bound to spores, and electron microscopy images all confirm that phages bind to P. larvae spores. Phage therapy for the treatment of AFB is an exciting avenue not only as an alternative to chemical antibiotics, but rather a treatment that can neutralize P. larvae spores.

<p>This dissertation is primarily focused on the <i>Buellia</i> species with pluriseptate spores, often referred to the section or genus <i>Diplotomma</i>, and secondarily on the Physciaceae. An ultrastructural study at family level gives new information on spore characters. Variations in the composition and structure of the spore wall layers and septa are described.</p><p>A phylogenetic analysis of the Physciaceae, based on morphological and chemical characters, is presented. In the resulting trees the species with pluriseptate spores appear in a poorly resolved clade together with other crustose species with 1-septate spores. A subsequent analysis, excluding foliose and fruticose species, shows that the species with pluriseptate spores do not form a monophyletic group, and that only a few are grouped together with the type species of <i>Diplotomma</i>. Due to low resolution, poor branch support, and uncertainty concerning the typification of <i>Buellia</i> all species treated are retained in <i>Buellia</i>.</p><p>Altogether 35 species with pluriseptate spores are treated, including ten new to science, viz. <i>B. aeruginosa</i>, <i>B. morsina</i>, <i>B. muriformis</i>, <i>B. oidaliella</i>, <i>B. pallido-marginata</i>, <i>B. romoletia</i>, <i>B. rubroreagens</i>, <i>B. terricola</i>, <i>B. tombadorensis</i>, and <i>B. tri-septata</i>. Historical outlines are presented, and information on life strategy, morphology, chemistry, ecology, and distribution is given. All species are similar and macroscopically not easily distinguished, having crustose thalli and blackish apothecia, but there is a great variation in microscopic characters, secondary chemistry, substrate preferences, habitat ecology, and distribution range. Several names are typified and many synonyms are listed. References are given to all species referred to <i>Diplotomma</i> in literature.</p>

This dissertation is primarily focused on the Buellia species with pluriseptate spores, often referred to the section or genus Diplotomma, and secondarily on the Physciaceae. An ultrastructural study at family level gives new information on spore characters. Variations in the composition and structure of the spore wall layers and septa are described. A phylogenetic analysis of the Physciaceae, based on morphological and chemical characters, is presented. In the resulting trees the species with pluriseptate spores appear in a poorly resolved clade together with other crustose species with 1-septate spores. A subsequent analysis, excluding foliose and fruticose species, shows that the species with pluriseptate spores do not form a monophyletic group, and that only a few are grouped together with the type species of Diplotomma. Due to low resolution, poor branch support, and uncertainty concerning the typification of Buellia all species treated are retained in Buellia. Altogether 35 species with pluriseptate spores are treated, including ten new to science, viz. B. aeruginosa, B. morsina, B. muriformis, B. oidaliella, B. pallido-marginata, B. romoletia, B. rubroreagens, B. terricola, B. tombadorensis, and B. tri-septata. Historical outlines are presented, and information on life strategy, morphology, chemistry, ecology, and distribution is given. All species are similar and macroscopically not easily distinguished, having crustose thalli and blackish apothecia, but there is a great variation in microscopic characters, secondary chemistry, substrate preferences, habitat ecology, and distribution range. Several names are typified and many synonyms are listed. References are given to all species referred to Diplotomma in literature.

Palynologic samples for this study were collected in April of 1975 at the Hawthorne and Minnehaha Mines near Linton and Dugger, Indiana, respectively. All samples were taken from the Coal No. 7 and the overlying shale.A total of eighty-three species assigned to thirty-one genera were recovered. Sixty-six species assigned to twenty-nine genera were recovered from the Hawthorne Mine and forty-seven species assigned to twenty-three genera were recovered from the Minnehaha Mine. Comparison of the palynomorph floras of the two localities reveals twenty-one genera and thirty-three species in common to both mines.Palynologic data from the sample area reveals two slightly different depositional basins. The ecology of the area was probably a coastal-swamp type of environment with an abundance of arborescent lepidodendrids.Ball State UniversityMuncie, IN 47306

Clostridium difficile is a common and potentially fatal cause of antibiotic-associated diarrhoea and pseudomembranous colitis worldwide. It has been isolated from patients and their surroundings, in healthcare facilities and from the community. C. difficile is able to survive for many months on inanimate surfaces in the form of spores. PCR ribotyping is used in the UK to characterise and identify strain diversity. Investigating how the most problematic strains respond to cleaning regimes may influence the control of disease. This work used the University Hospitals of Leicester Trust as a case study for this purpose of understanding the epidemiology of this pathogen within healthcare facilities. Five individual agar media were compared based on their abilities to recover and resuscitate damaged ribotype 027 spores, a strain associated with disease outbreaks and increased severity. Controlled laboratory experiments with a sub-lethal dose of a germicide were conducted before C. difficile recovery from hospital wards. An additional two sampling campaigns acquired environmental strains. C. difficile isolation after routine cleaning demonstrated the inefficiency of the current recovery regime as C. difficile spores were recovered using direct contact plates, enrichment broths, and resuscitation media. This study used layering of non-selective agar over selective agar, identifying a potential link in the proportions of media following the use of sponges in environmental sampling. All strains were characterised by ribotyping; ribotype 027 was isolated from all sampling cohorts. A four-month epidemiological study was conducted into the ribotype prevalence and distribution from C. difficile-positive faecal specimens. A second survey investigated these effects with a modification of C. difficile detection from faecal samples. Hydrogen peroxide vapour is currently being explored as a means of decontamination of healthcare-associated infections. Inactivation kinetics of ribotype 027 spores were analysed in response to vapour and liquid exposure of hydrogen peroxide. No reports thus far have explored such kinetics and controlled decontamination with both clinical and non-clinical strains. Evidence strongly suggests spores can be inactivated with its application. Furthermore, this study revealed there appears to be significant differences in susceptibility and inactivation of different C. difficile ribotypes.