Os produtos naturais desempenham fundamental importância na descoberta de novos fármacos. Muitos destes fármacos originaram-se de produtos naturais microbianos o que demonstra uma extrema eficiência na habilidade dos micro-organismos de sintetizar substâncias químicas com elevado potencial biológico. Sabe-se que os insetos sociais estão sujeitos a condições climáticas e populacionais que aumentam sua susceptibilidade a parasitas. Assim, estes insetos desenvolveram um mecanismo de defesa evolutivo que consiste na associação simbiótica a bactérias capazes de biossintetizar produtos antimicrobianos contra os patógenos. Dispondo dessas informações, o presente trabalho contribuiu com o estudo dos produtos naturais biossintetizados pela bactéria Paenibacillus polymyxa ALLI-03-01, provável simbionte da espécie de abelha sem ferrão Melipona scutellaris. Os micro-organismos foram isolados de colônias mantidas no Departamento de Biologia da FFCLRP-USP. Todas as linhagens foram testadas contra dois fungos entomopatogênicos - Beauveria bassiana e Metarhizium anisopliae; os que apresentaram atividade antifúngica tiveram seus extratos em acetato de etila e metanol preparados e foram avaliados em ensaios antiparasitários contra Trypanosoma cruzi e Leishmania donovani. A bactéria ALLI-03-01, isolada do alimento larval, foi escolhida para os estudos químicos, pois apresentou satisfatória atividade antifúngica e proeminente atividade antiparasitária. Este micro-organismo foi identificado pelo sequenciamento do gene 16S rRNA como sendo a bactéria Paenibacillus polymyxa, conhecida pela alta capacidade de produção de metabólitos secundários. Para conhecer seu metabolismo, esta bactéria foi cultivada em meio ISP-2 ágar e líquido e do extrato em acetato de etila do cultivo fermentativo de P. polymyxa ALLI-03-01 foi isolado o ácido L-3-fenil lático. As atividades antifúngicas dos padrões comerciais dos ácidos D-(+)- e L-(-)- 3-fenil lático foram testadas frente ao fungo entomopatogênico B. bassiana e apenas o enantiômero L apresentou atividade antifúngica. Do extrato em metanol do cultivo em meio sólido do mesmo micro-organismo foram identificados em mistura, principalmente por MALDI-TOF MS/MS, nove diferentes ciclolipodepsipeptídeos, da classe das Fusaricidinas. As fusaricidinas são conhecidas por apresentarem alta atividade anti-microbiana, que foi comprovada quando duas frações compostas por diferentes fusaricidinas foram testadas frente ao fungo entomopatogênico Metarhizium anisopliae e contra a bactéria patogênica Paenibacillus larvae. As frações apresentaram resultados satisfatórios de inibição contra ambos os patógenos, dessa maneira as fusaricidinas e a produção seletiva do ácido L-3-fenil lático por esta bactéria podem ser vias de proteção da colônia, evitando que patógenos destruam-na e permitindo o desenvolvimento sadio de larvas. Sendo assim, este trabalho relata pela primeira vez a possível relação simbiótica existente entre o micro-organismo Paenibacillus polymyxa ALLI-03-01 e a colônia de abelhas Melipona scutellaris, bem como a produção por este micro-organismo de compostos com alta atividade antimicrobiana que podem exercer o controle de parasitas dentro da colônia, estabelecendo assim, uma possível relação ecológica entre o micro-organismo destacado e as abelhas Melipona scutellaris. / Natural products play an important role in drug discovery. Several drugs have been discovered or developed from microbial natural products hits and leads showing a great efficiency of microorganisms to synthesize compounds with high biological potential. It is known that social insects are subjected to climate and population conditions that increase their susceptibility to parasites. Therefore, these insects have developed an evolutionary defense mechanism consisting in symbiotic association with bacteria capable of biosynthesize antimicrobial products against pathogens. In this context, this study contributed to the knowledge of natural products biosynthesized by the bacterium Paenibacillus polymyxa ALLI-03-01, a probable symbiont of the stingless bee Melipona scutellaris. Microorganisms were isolated from colonies maintained at the Department of Biology of FFCLRP-USP. All strains were tested against two entomopathogenic fungi - Beauveria bassiana and Metarhizium anisopliae; strains that showed antifungal activity had their ethyl acetate and methanol extracts prepared and evaluated against the parasites Trypanosoma cruzi and Leishmania donovani. The bacterium ALLI-03-01, isolated from larval food, was selected for chemical studies, due to its satisfactory antifungal activity and outstanding antiparasitic activity. This microorganism was identified by gene 16S rRNA sequencing as Paenibacillus polymyxa, a bacterium recognized for its ability of biosynthesize secondary metabolities. This bacterium was cultivated in ISP-2 agar and liquid medium and from the ethyl acetate extract of P. polymyxa ALLI-03-01 cultivated in liquid medium L-3-phenyllactic acid was isolated. The antifungal activity of comercial standards of D-(+)- and L-(-)-phenyllactic acid were tested against entomopathogenic fungus B. bassiana and only the L enantiomer presented antifungal activity. Nine cyclo lipodepsipeptides known as fusaricidins were identified in mixture using MALDI-TOF-MS/MS from the methanol extract of ALLI-03-01. The fusaricidins are recognized for their high antimicrobial activity, which was confirmed when two fractions composed by different fusaricidins were tested against the entomopathogenic fungus Metarhizium anisopliae and the pathogenic bacterium Paenibacillus larvae. The fractions showed satisfactory inhibition against both pathogens. Therefore, fusaricidins and selective production of L-3-phenyllactic acid by the bacterium may be colony protection resources, preventing the action of pathogens and allowing healthy larvae development. Therefore, this study reports for the first time a possible symbiotic relationship between Paenibacillus polymyxa ALLI-03-01 and the colony of M. scutellaris bees, as well as production of compounds with high antimicrobial activity by this microorganism that might act as parasite control agents within the colony, establishing a possible ecological relationship between the bacterium and M. scutellaris bees.

In this thesis, the interactions of U(VI) with one representative each of the domains Bacteria (Paenibacillus sp. JG TB8) and Archaea (Sulfolobus acidocaldarius) are compared. We demonstrate that at highly acidic conditions (pH ≤ 3), U(VI) is bound to cells of the both strains exclusively via organic phosphate groups. In contrast to this, the U(VI) complexation modes differ between the studied strains at moderate acidic conditions. These differences are assigned to the different cell wall structures of both strains as well as to their different physiological characteristics. We also demonstrate that the aeration conditions can strongly influence the uranium accumulation of facultative anaerobic microorganisms at moderate acidic pH conditions. This finding could clearly be assigned to the dependency of the intrinsic phosphatase activity on the aeration conditions. The second part of this thesis deals with the outermost surface layer (SlaA-layer) of S. acidocaldarius. It was shown that this surface protein is not involved in the U(VI) complexation at highly acidic conditions, covering the physiological pH optimum of S. acidocaldarius. Hence the SlaA layer does not provide a protective function against U(VI) to the cells of this acidophilic archaeon. However, we demonstrated that purified SlaA-layer ghosts (i.e. empty cell sacculi) efficiently interact with gold ions and are a good macromolecular template for the formation of magnetic gold nanoparticles. / In dieser Doktorarbeit werden die Wechselwirkungen von U(VI) mit je einem Vertreter der Bakterien (Paenibacillus sp. JG TB8) und Archeen (Sulfolobus acidocaldarius) verglichen. Wir konnten zeigen, dass U(VI) im sehr sauren Milieu (pH ≤ 3) ausschließlich durch organische Phosphatgruppen an die Zellen beider Stämme gebunden ist. Im Gegensatz dazu unterscheiden sich die Mechanismen der U(VI)-Komplexierung beider untersuchter Stämme bei mäßig sauren Bedingungen voneinander. Diese Unterschiede basieren auf den unterschiedlichen Zellwandstrukturen und physiologischen Eigenschaften beider Stämme. Wir konnten außerdem zeigen, dass die atmosphärischen Bedingungen die Urankomplexierung durch fakultativ anaerobe Mikroorganismen bei mäßig sauren Bedingungen stark beeinflussen kann. Dieses Ergebnis konnte eindeutig auf die von den atmosphärischen Bedingungen-abhängige, enzymatische Aktivität der zelleigenen Phosphatase zurückgeführt werden. Der zweite Teil dieser Arbeit beschäftigt sich mit der äußeren Oberflächenschicht (SlaA-layer) von S. acidocaldarius. Es konnte gezeigt werden, dass dieses Oberflächenprotein nicht an der U(VI)-Komplexierung bei stark sauren pH, welcher dem physiologischen pH Optimum von S. acidocaldarius entspricht, beteiligt ist. Damit stellt der SlaA-layer keinen Schutz gegen Uran für die Zellen dieses azidothermophilen Archaeons dar. Allerdings konnten wir zeigen, dass isolierte „SlaA-layer ghosts“ (d.h. leere Zellhüllen) mit Goldionen interagieren und sich daher als makromolekulares Template für die Herstellung magnetischer Gold Nanopartikel eignen.

4 Summary Altogether, the six most common bee viruses which infect the honey bee (Apis mellifera) were monitored in the territory of the Czech Republic between 2006 and 2009. Parallel infections of viruses (DWV, ABPV and BQCV) in bee adults and parallel co- infection of viruses with fungal diseases caused by Nosema apis and Nosema ceranae were confirmed by PCR tests. A new sensitive method of detection of the originator of the American foulbrood (Paenibacillus larvae) from bee debris was developed for the practical use of detection of AFB disease in bee populations. Various approaches for the extraction of spores from bee debris and lyses of spores were compared. The sensitivity of PCR tests for the presence of Paenibacillus larvae in debris was compared with the classic cultivation method. The PCR method for the detection American foulbrood was further studied and developed to be more efficient. A new method, based on a matrix-like sample re-arrangement and a use of pooled samples, has been developed for testing 1000 samples in 35 PCR reactions. Another goal was to develop a robust and fast screening method for American foulbrood based on the cultivation test using paper sheets RIDA®COUNT with a specific cultivation medium, specific selection conditions for Paenibacillus larvae and chromogen visualization...

Submitted by GUILHERME DEOMEDESSE MINARI null (gdminari@gmail.com) on 2016-03-02T21:34:04Z No. of bitstreams: 1 Dissertação Guilherme D. Minari.pdf: 1275061 bytes, checksum: b7c17ca0d2c40870526f019d55c9b421 (MD5) / Rejected by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: A versão do arquivo submetida é considerada a versão final do trabalho, não sendo possível realizar alterações posteriormente. Por favor verifique as páginas que contém a ficha catalográfica e o certificado de aprovação e realize uma nova submissão certificando-se de que o arquivo PDF contém a versão final do trabalho. Agradecemos a compreensão. on 2016-03-04T13:14:40Z (GMT) / Submitted by GUILHERME DEOMEDESSE MINARI null (gdminari@gmail.com) on 2016-03-04T18:31:39Z No. of bitstreams: 2 Dissertação Guilherme D. Minari.pdf: 1275061 bytes, checksum: b7c17ca0d2c40870526f019d55c9b421 (MD5) Dissertação_Guilherme_Deomedesse_Minari.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-04T19:56:27Z (GMT) No. of bitstreams: 1 minari_gd_me_jabo.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) / Made available in DSpace on 2016-03-04T19:56:27Z (GMT). No. of bitstreams: 1 minari_gd_me_jabo.pdf: 1505672 bytes, checksum: 206338224f2d6ca316c769acb346fc78 (MD5) Previous issue date: 2016-02-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A contaminação do solo pode ser decorrente do descarte inadequado de substâncias com potencial poluente ou do uso agrícola prolongado com aplicação de materiais corretivos de acidez, fertilizantes e defensivos. O objetivo deste estudo foi avaliar a distribuição espacial de cádmio (Cd), cromo (Cr) e níquel (Ni) em Latossolo sob plantio direto, convencional e convencional com aplicação de lodo de esgoto, em áreas com exploração agrícola há mais de 80 anos. As concentrações dos metais em 422 amostras de solo foram mensuradas por espectroscopia de absorção atômica com chama ar-acetileno, sendo os extratos obtidos por extração com HNO3, H2O2 e HCl em sistema aberto (USEPA-3050B). As variáveis pH e matéria orgânica também foram determinadas. Os mapas de distribuição espacial foram elaborados a partir do software Spring v. 5.2.7 e auxiliaram na visualização da dispersão dos metais na área de estudo, indicando níveis de contaminação. A análise estatística multivariada por componentes principais diferenciou as amostras de solo de mata nativa das que estão sob atividade agrícola, apontando a uma mesma fonte de contaminação para Cr e Ni, como também para a correlação da matéria orgânica com Cd. Conclui-se que a área estudada apresenta contaminação, em larga escala, por Cd e Cr, e contaminações pontuais por Ni, estando alguns valores dentro da faixa de intervenção. / Soil contamination may result from inappropriate use of substances with pollution potential or from the prolonged agricultural use with application of acidity corrective materials, fertilizers and pesticides. The aim of this work was to evaluate the spatial distribution of cadmium (Cd), chromium (Cr) and nickel (Ni) in latosol under no tillage, conventional tillage and conventional with sewage sludge application, in agricultural exploration areas for over 80 years. Metals concentration in 422 soil samples were measured by atomic absorption spectroscopy with acetylene-air flame, obtaining the extracts by extraction with HNO3, H2O2 e HCl in open system (USEPA-3050B). The variables pH and organic matter were obtained. Maps of spacial distribution were elaborated by software Spring v. 5.2.7 and assisted in the visualization of the metals dispersion in the study area, indicating contamination levels. The statistical multivariate by main components differentiated the native forest soil samples from those under agricultural activity, pointing to a same contamination source to Cr and Ni, and to the organic matter correlation to Cd as well. It can be concluded that the studied area presents contamination in large scale by Cd and Cr and pontual contamination by Ni, being some of the values into the intervention group.

The Paenibacillaceae family of bacteria includes two species known to infect the hives of honeybees, Paenibacillus larvae and Brevibacillus laterosporus. P. larvae, the causative agent of American Foulbrood (AFB) causes a lethal infection of honeybee larvae, while B. laterosporus is a secondary invader following European Foulbrood (EFB) infection. Increasing antibiotic resistance of P. larvae bacteria has prompted a search for alternative treatment methods for this disease. Bacteriophages are the most diverse life forms on earth and can provide important insights about the bacterial hosts they infect. However, few Paenibacillaceae phages have been isolated or characterized. In this study, the first B. laterosporus phages are characterized with respect to host range, structural morphology, and sequence similarity. The isolation and characterization of many P. larvae field isolates together with 38 novel P. larvae phages made possible the first broad phage typing study of P. larvae. Phage typing data indicated that P. larvae strains tested could be categorized into one of two groups. Comparative genomics of bacteriophages was made easier by modifying Phamerator to make it broadly accessible and usable to phage researchers throughout the world. Additionally, raw sequencing data can now be used to identify phage DNA packaging strategies that are indicative of a phage’s physical ends. Using these data, phage genomes can be published in an orientation and complementarity that reflects the physical structure of the phage chromosome, providing order and consistency that will benefit all future phage researchers.

This diploma thesis deals with a serious honey bee (Apis mellifera) disease, the American foulbrood (AFB), and with possibilities of its control. The thesis is divided into two parts, the theoretical and the experimental. The theoretical part is written as a research and contains two big chapters. The first one describes American foulbrood disease and its causative agent, bacteria Paenibacillus larvae. The second part deals with the wax moth (Galleria mellonella). The experimental part then combines above mentioned themes in an effort to find the relationship between the wax moth caterpillars and bacteria P. larvae that could possibly help to control the AFB. The aim of this work was to determine whether the wax moth can disrupt resistant layers of P. larvae spores thanks to its well adapted digestive tract and whether it could change their germination.

Made available in DSpace on 2014-06-11T19:35:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-11-30Bitstream added on 2014-06-13T20:06:05Z : No. of bitstreams: 1 bernardo_era_dr_botfca.pdf: 350867 bytes, checksum: 5ac2bf8356b892e6a8597025678f2c2e (MD5) / O Brasil é o maior produtor de laranja e o maior exportador mundial de suco concentrado. A pinta preta dos citros, causada por Guignardia citricarpa, é uma doença de grande importância econômica, principalmente para o Estado de São Paulo. O interesse no controle biológico de fitopatógenos, como alternativa de controle e como forma de reduzir os problemas ocasionados pelo uso intensivo de fungicidas, tem levado ao desenvolvimento de técnicas alternativas para uma agricultura mais sustentável. O trabalho teve por objetivo avaliar o efeito de agentes de controle biológico (Bacillus subtilis e Trichoderma harzianum) e produtos alternativos (biofertilizantes e leite) no controle da pinta preta dos frutos cítricos em cultivos convencional e orgânico. Além disso, foi avaliado o efeito de Bacillus subtilis, Paenibacillus lentimorbus e Streptomyces sp. no controle de G. citricarpa e Penicillium digitatum em pós-colheita. Os experimentos em campo foram realizados em pomar de laranja`Valência´ e `Pêra´, localizadas nos municípios de Conchal e Santa Eudóxia, SP, respectivamente. Em cultivo convencional de `Valência´ foram avaliados dois biofertilizantes (Microgeo® e Bio2), nas safras 2003/2004 e 2004/2005, com 15 repetições por tratamento, sendo uma planta por repetição. As árvores foram pulverizadas em intervalos de 28 dias, sendo o início em 08/12/03, para a safra 2003/2004 e 08/11/2004, para a safra 2004/2005. Na safra 2003/2004 as concentrações utilizadas do biofertilizante Microgeo® foram 0; 10; 20; 30 e 40%. Na safra 2004/2005 foram repetidos os mesmos tratamentos dasafra anterior e incluído tratamento com o biofertilizante Bio2 nas concentrações de 0; 2,5; 5,0; 7,5 e 10%. Para as avaliações foram utilizadas uma escala de notas de 1=0,5% a 6=49% da área do fruto atacada. Os biofertilizantes apresentaram comportamento... / Brazil is the worldwide biggest producer and exporter of orange and orange juice. The citrus black spot (CBS), caused by Guignardia citricarpa, is a disease of great economic importance, mainly for the São Paulo State. The interest in biological control of plant pathogens, as mitigation of the problems caused by intensive use of fungicides, has led to development of alternative techniques for a more sustainable agriculture. The objective of this work was to evaluate the effects of biocontrol agents (Bacillus subtilis and Trichoderma sp.) and other biocompatible products (cow milk and biofertilizers) for managing CBS in organic and conventional systems. Besides, the effect of B. subtilis, Paenibacillus lentimorbus and Streptomyces sp. for control of G. citricarpa and Penicillium digitatum, in post-harvest was also evaluated. The filed experiments were carried through in trees of `Valencia' and `Pera´, located in Conchal and Santa Eudóxia, SP, respectively. In `Valencia´ conventional system were evaluated two biofertilizers (Microgeo® and Bio2). The experiments were conducted at harvests 2003/2004 and 2004/2005. The trees were sprayed in 28 days intervals, and the first was in 08 December 2003 in 2003/2004 harvest, and 08 November 2004 in 2004/2005 harvest.

Orientador: Wagner Bettiol / Banca: Antonio Carlos Maringoni / Banca: Edson Luiz Furtado / Banca: Antonio de Goes / Banca: Katia Cristina Kupper Moretto / Resumo: O Brasil é o maior produtor de laranja e o maior exportador mundial de suco concentrado. A pinta preta dos citros, causada por Guignardia citricarpa, é uma doença de grande importância econômica, principalmente para o Estado de São Paulo. O interesse no controle biológico de fitopatógenos, como alternativa de controle e como forma de reduzir os problemas ocasionados pelo uso intensivo de fungicidas, tem levado ao desenvolvimento de técnicas alternativas para uma agricultura mais sustentável. O trabalho teve por objetivo avaliar o efeito de agentes de controle biológico (Bacillus subtilis e Trichoderma harzianum) e produtos alternativos (biofertilizantes e leite) no controle da pinta preta dos frutos cítricos em cultivos convencional e orgânico. Além disso, foi avaliado o efeito de Bacillus subtilis, Paenibacillus lentimorbus e Streptomyces sp. no controle de G. citricarpa e Penicillium digitatum em pós-colheita. Os experimentos em campo foram realizados em pomar de laranja'Valência' e 'Pêra', localizadas nos municípios de Conchal e Santa Eudóxia, SP, respectivamente. Em cultivo convencional de 'Valência' foram avaliados dois biofertilizantes (Microgeo® e Bio2), nas safras 2003/2004 e 2004/2005, com 15 repetições por tratamento, sendo uma planta por repetição. As árvores foram pulverizadas em intervalos de 28 dias, sendo o início em 08/12/03, para a safra 2003/2004 e 08/11/2004, para a safra 2004/2005. Na safra 2003/2004 as concentrações utilizadas do biofertilizante Microgeo® foram 0; 10; 20; 30 e 40%. Na safra 2004/2005 foram repetidos os mesmos tratamentos dasafra anterior e incluído tratamento com o biofertilizante Bio2 nas concentrações de 0; 2,5; 5,0; 7,5 e 10%. Para as avaliações foram utilizadas uma escala de notas de 1=0,5% a 6=49% da área do fruto atacada. Os biofertilizantes apresentaram comportamento ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Brazil is the worldwide biggest producer and exporter of orange and orange juice. The citrus black spot (CBS), caused by Guignardia citricarpa, is a disease of great economic importance, mainly for the São Paulo State. The interest in biological control of plant pathogens, as mitigation of the problems caused by intensive use of fungicides, has led to development of alternative techniques for a more sustainable agriculture. The objective of this work was to evaluate the effects of biocontrol agents (Bacillus subtilis and Trichoderma sp.) and other biocompatible products (cow milk and biofertilizers) for managing CBS in organic and conventional systems. Besides, the effect of B. subtilis, Paenibacillus lentimorbus and Streptomyces sp. for control of G. citricarpa and Penicillium digitatum, in post-harvest was also evaluated. The filed experiments were carried through in trees of 'Valencia' and 'Pera', located in Conchal and Santa Eudóxia, SP, respectively. In 'Valencia' conventional system were evaluated two biofertilizers (Microgeo® and Bio2). The experiments were conducted at harvests 2003/2004 and 2004/2005. The trees were sprayed in 28 days intervals, and the first was in 08 December 2003 in 2003/2004 harvest, and 08 November 2004 in 2004/2005 harvest. / Doutor

Despite an increase of biotechnical studies in third world countries, the bioinformatical side is largely lacking. In this paper we attempt to further the bioinformatical capabilities of east Af-rica. The project consisted of two teaching segments for east African doctorates, one as part of an academic workshop at ILRI, Kenya, and one in a small class at SLU, Sweden. The project also included the generation of two simple to use bioinformatical pipelines with the explicit aim to be reused by novice bioinformaticians from the very same region. The viability of the piplines were verified by generating transcriptional expression level differences for Paeni-bacillus polymyxa strain A26 and whole genome annotations for Setaria digitata. Both pipe-lines may have some merit for the collaborative effort between ILRI and SLU to annotate Eleusine coracana, a draught resilient crop, the annotation of which may save lives. The teaching material, source code for the pipelines and overall teaching impression have been included in this paper.

Paenibacillus larvae is a gram-positive spore-forming bacterium that affects and kills the larvae of the honey bee (Apis mellifera) and causes the American foulbrood disease. Adults bees do not become infected, but they transmit tenacious spores within the hive and between hives and can infect larvae while caring for them. It is not allowed by law to treat bees in the Czech Republic, but the recommended preventive method for reducing the amount of spores in the hive is the shook swarm method (bees are moved to a new clean hive and the old hive is destroyed with all brood and supplies). The aim of this work was to detect and quantify P. larvae in bee workers using the quantitative real-time polymerase chain reaction (qPCR). In the first experiment, the two set of samples were taken - bees before and after the shook swarm method, but the expected decrease in spores in the samples taken after shook swarm was not confirmed, and conversely, non-specific products were amplified. In the second experiment, the presence of P. larvae spores in samples from heavily infected hives (with clinical symptoms of American foulbrood) and from hives with almost no findings of P. larvae spores, both originating from the same habitat, were compared. In this case, the differences were clearly visible. There were not...