Interactions are all around us, and as humans we may use words and gestures to communicate our intentions. At the micro level of fungi, communications are replaced by chemical signals and structure. These interactions fall into three distinctive categories: synergistic, where organisms help each other, as is the case with ectomycorrhizal fungi and tree roots, deadlock, or combat, where organisms fight for or defend a resource. When it comes to fungi-tree interactions, the fungi group of basidiomycetes fall into the latter category. At the onset of fungal infection, a living tree defends itself by producing resinous substances such as terpenes. These compounds are frequently found in hydrodistilled turpentine, which makes turpentine a prime source of antifungal compounds. A D-optimal design of fractionated turpentine together with gas chromatography (GC) coupled to a mass spectrometer was employed to find the most biologically active constituent of turpentine. Growth rate of Coniophora puteana was used to assess the efficacy of the mixed fractions. The partial least squares projection model had an excellent predictive power (R2 = 0.988, Q2 = 0.825) and validity. A putative sesquiterpene was identified as the most active compound for inhibiting fungal growth. The model was corroborated by an external validation assay employing preparative GC. After the death of a tree, fungi are no longer hindered by secondary metabolites from the tree. Instead, other interspecies interactions and intraspecies interactions, such as fungi-fungi interactions, occur. We found that when the white-rot fungus Heterobasidion parviporum and brown-rot fungus Gloeophyllum sepiarium interact with each other, amino acids are used to a higher extent. Amino acids may be used to produce antifungal compounds to hinder the other species from growing. Lysine in particular was utilized to a greater extent during interaction. Glutamine was the only amino acid that increased in concentration. Glutamine might be exuded or converted by enzymes from already existing glutamic acid. Dry weights suggest that the fungi were in a deadlock and that nutrient limitation might be a determining factor. It seemed that H. parviporum was favoured by a decrease in pH while the opposite pattern may be true for G. sepiarium.

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.

En aquesta tesi s'han dut a terme dos tipus d'estudis diferents. L'objectiu del primer era la preservació del semen de porcí a 15ºC i el del segon eren els co-cultius homòlegs de cèl·lules epitelials de l'oviducte i espermatozoides de porcí. Pel que fa al primer estudi, s'ha observat que l'addició de la prostaglandina F2α i àcid hialurònic a les dosis seminals no malmena la qualitat espermàtica i que la tolerància dels espermatozoides als canvis d'osmolalitat del medi es pot correlacionar proves de fertilitat i prolificitat..Respecte el segon, s'ha determinat que les cèl·lules oviductals afecten els paràmetres espermàtics i que la presència d'espermatozoides sobreexpressa els gens que codifiquen per les proteïnes de xoc tèrmic. Així, se suggereix que aquestes proteïnes tenen algun paper en els processos reproductius que tenen lloc a l'oviducte, malgrat que s'hagi observat, mitjançant la tècnica de la interferència de l'RNA, que la HSP90AA1 no està implicada en el perllongament de la viabilitat espermàtica. / In this thesis, two different studies have been conducted. The aim of the first experimental chapter was boar sperm preservation at 15ºC, the second dealing with in vitro homologous co-culture of oviductal epithelial cells (OEC) and spermatozoa. Regarding the first, it has been observed that the addition of prostaglandin F2α and hyaluronic acid do not cause any harm on sperm quality, and the osmotic tolerance of spermatozoa can be correlated with fertility and prolificacy rates of a given ejaculate.As far as the second study is concerned, OEC specifically affect sperm functional parameters and the presence of spermatozoa upregulates the expression of some genes encoding for heat shock proteins. Some role in the reproductive processes taking place in the oviduct is therefore suggested for this protein family, even though it has been observed, by means of RNA interference, that HSP90AA1 is not the protein involved in prolonging sperm survival.

Solid substrate cultivation (SSC) using lignocellulosic non-food feedstock, such as switchgrass, is an alternative for advanced biofuel production. Acetone-Butanol-Ethanol (ABE) fermentation in two stages using a sequential culture of microorganisms from the class Clostridia is an approach proposed to increase the butanol production. The goal was to test the efficacy of a sequential culture on high solid substrate cultivation in batch and continuous cultivation, and to evaluate conditions to optimize butanol production using switchgrass as substrate. Initial batch experiments were used to determine particle size effect, choice of solvent producer and pretreatment evaluation: The effect of particle size on gas production was surface area-dependent, 2 mm particle size of switchgrass was better fermented by clostridia than the other particle sizes. C. thermocellum improved switchgrass fermentation by C. beijerinckii. Moreover, C. saccharoperbutylacetonicum produced the highest butanol yield on glucose as substrate. The Fenton reaction was studied as a potential pretreatment for switchgrass. C. beijerinckii grew better on Fenton-treated material, but solvent production was low. The major conclusion of the continuous flow on SSC experiment was that there is no statistical difference in the effect of flow rate within the flow range tested.

Submitted by ROBERTH NASCIMENTO DA TRINDADE null (octuberman@gmail.com) on 2016-02-02T12:22:18Z No. of bitstreams: 1 ROBERTH TRINDADE - Exploração racional da rizosfera de Senna spectabilis - interações entre as bactérias Pseudoxanthomonas indica e Shigella sp..pdf: 2399531 bytes, checksum: 1d992c19e1d2db2f229774b0f45cc074 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-03T17:44:59Z (GMT) No. of bitstreams: 1 trindade_rn_me_araiq_par.pdf: 740045 bytes, checksum: 46f1e680a7415dd924948bb180d6dfc7 (MD5) / Made available in DSpace on 2016-02-03T17:44:59Z (GMT). No. of bitstreams: 1 trindade_rn_me_araiq_par.pdf: 740045 bytes, checksum: 46f1e680a7415dd924948bb180d6dfc7 (MD5) Previous issue date: 2016-01-22 / O descobrimento de novas moléculas bioativas tem sido o grande alvo ao decorrer dos anos da química dos produtos naturais, particularmente, micro-organismos tem se evidenciado como uma importante fonte do descobrimento de novos fármacos. Uma ampla gama de compostos provenientes de organismos microbianos são relatados na literatura, porém, recentes pesquisas demonstram que tais micro-organismos podem sofrer indução por determinados agentes externos, químicos ou físicos, através de mecanismos de defesa ou até mesmo o mutualismo existente em determinado meio, produzindo novas estruturas químicas, que até então, tinham sua rota biosintética silenciada devido a metodologias padrões de cultivo isolado. Através de utilização de culturas mistas de bactérias, o presente trabalho proporciona o conhecimento da relação existente entre Pseudoxanthomonas indica e Shigella sp., ambas provenientes da rizosfera de Senna spectabilis. Para isso, as bactérias foram cultivadas em meio de cultivo líquido czapek broth, sendo realizado o estudo do crescimento das bactérias e produção de metabolitos frente ao tempo, tendo em vista uma maximização de resultados e maior indução de compostos químicos. A utilização de ferramentas analíticas de análise rápida e eficiente como a cromatografia líquida de alta eficiência (HPLC) associada à espectrometria de massas de alta resolução (HRMS) propiciaram a obtenção do perfil cromatográfico dos micro-organismos estudados em cultura mista e simples, sendo possível observar a existência de compostos químicos produzidos apenas em co-cultura, evidenciado o potencial existente da abordagem utilizada. / The discovery of new bioactive molecules has been the big target to over the years the chemistry of natural products, particularly micro-organisms has been shown to be an important source of discovery of new drugs. A wide range of compounds from microbial organisms are reported in the literature, however, recent research shows that these micro-organisms can undergo induction by certain external, chemical or physical agents, through defense mechanisms or even the existing mutualism on a particular medium, producing new chemical structures, which until then had silenced their biosynthetic route due to methodologies patterns of isolated farming. Through use of mixed cultures of bacteria, this study provides knowledge of the relationship between Pseudoxanthomonas indica and Shigella sp., Both from the rhizosphere of Senna spectabilis. For this, bacteria were grown in liquid Czapek broth cultivation being conducted the study of the growth of bacteria and production of metabolites against time, with a view to maximizing results and greater induction of chemical compounds. The use of analytics quickly and efficiently analyzes such as high-performance liquid chromatography tools (HPLC) associated with high resolution mass spectrometry (HRMS) enabled the obtaining of the chromatographic profile of microorganisms studied in mixed and single culture, and you can to observe the existence of chemical compounds produced only in co-culture demonstrated the potential of the approach used.

Submitted by Carolina Rabal Biasetto null (carolinarabal@yahoo.com.br) on 2016-05-18T19:36:08Z No. of bitstreams: 1 Tese_Doutorado_Carolina_Rabal_Biasetto_2016.pdf: 15348096 bytes, checksum: 59245cf961f6aaf1a999156f53853b5d (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-05-20T18:01:57Z (GMT) No. of bitstreams: 1 biasetto_cr_dr_araiq_par.pdf: 1896284 bytes, checksum: 8b4dcb25761fe239f657abc24e10c1e7 (MD5) / Made available in DSpace on 2016-05-20T18:01:57Z (GMT). No. of bitstreams: 1 biasetto_cr_dr_araiq_par.pdf: 1896284 bytes, checksum: 8b4dcb25761fe239f657abc24e10c1e7 (MD5) Previous issue date: 2016-04-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A capacidade biossintética dos fungos endofíticos, aliado aos estudos químico e biológicos relatados para Eugenia brasiliensis, motivou a idealização do projeto, a prospecção química, biológica e enzimática em fungos endofíticos associados a folhas, caules e frutos saudáveis de Eugenia brasiliensis e a avaliação do co-cultivo e epigenética em Xylaria cubensis, Diaporthe sp. e Colletotrichum sp., na obtenção de novas substâncias. O isolamento dos fungos endofíticos, resultou em dezessete linhagens de fungos endofíticos, sendo estes cultivados em escala reduzida em meio líquido de batata e dextrose (PDB) e Czapek, a 25 oC, sob modo estático para obtenção dos respectivos extratos brutos em AcOEt. A avaliação metabólica destes extratos foi realizada por CCDC, HPLC-DAD e RMN de 1H, como também a potencialidade enzimática e biológica pela avaliação das atividades antifúngica, anticolinesterásica e citotóxica, sendo que estes extratos demonstraram ser promissores. A prospecção inicial conduziu a seleção de três fungos endofíticos identificados como Xylaria cubensis (Eb_caH_5), Diaporthe sp. (Eb_caS_4), e Colletotrichum sp. (Eb_frmH_1), os quais foram cultivados (escala ampliada) em PDB para isolamento e determinação/elucidação estrutural dos metabólitos secundários. O estudo de Xylaria cubensis, resultou no isolamento de 8 substâncias, sendo da classe dos nucleosídeos, dicetopirazinas, isocumarinas e citocalasinas. De Diaporthe sp. foi isolado e identificado 8 substâncias: duas dicetopiperazinas, ácido nitropropiônico, uracila, tirosol, zygosporina D, pirrolidona (inédita) e alternariol. Colletotrichum sp. resultou no isolamento de 6 substâncias, sendo três dicetopiperazinas, além das substâncias N-(2-feniletil)acetamida, N-acetiltriptamina e metanoato de 2-hidroxibutila 3-indol (inédita). Todas as classes de substâncias produzidas por estes fungos endofíticos apresentam diversas atividades biológicas relatadas na literatura. Destaca-se a atividade fitotóxica da citocalasina D frente a coleóptilos de trigo superior ao herbicida comercial GOAL®. Para verificar a influência na produção metabólica de Xylaria cubensis, Diaporthe sp. e Colletotrichum sp., utilizou-se estratégias como o co-cultivo em meio sólido (PDA) e líquido (PDB) e a epigenética (escala reduzida), nas quais os endófitos mostraram produção metabólica diferente em relação à produção das monoculturas e na ausência do modulador epigenético ácido hidroxâmico suberoilanilida (SAHA), respectivamente. As ferramentas estatísticas, como PCA (Análise de componentes principais) e PLS-DA (Análise discriminante com calibração multivariada por mínimos quadrados parciais) permitiram uma rápida identificação e localização dos perfis metabólicos dos co-cultivos comparados às monoculturas. Estes dados contribuem para o conhecimento dos perfis metabólicos, biológicos e enzimáticos dos fungos endofíticos isolados de Eugenia brasiliensis, bem como suas interações com o hospedeiro. / The biosynthetic capacity of endophytic fungi, allied chemical and biological studies reported to Eugenia brasiliensis motivated the idealization of the project, prospection chemical, biological and enzymatic in endophytic fungi associated with healthy leaves, stems and fruits from Eugenia brasiliensis and evaluation co-culture and epigenetic in Xylaria cubensis, Diaporthe sp. and Colletotrichum sp., in obtaining new substances. The isolation of endophytic fungi resulted in seventeen endophytics fungi, these being cultivated in small scale on potato dextrose broth (PDB) and Czapek at 25 °C under static mode to obtain the corresponding crude extracts in AcOEt. The metabolic evaluation of these extracts was performed by CCDC, HPLC-DAD and 1H NMR, also enzymatic and biological potential were performed by antifungal, cytotoxic, and anticholinesterase activities, and these extracts have shown promise. The preliminary prospection led to the selection of three endophytic fungi identified as Xylaria cubensis (Eb_caH_5), Diaporthe sp. (Eb_caS_4) and Colletotrichum sp. (Eb_frmad_1) which were cultivated (larger scale) in PDB for isolation and determination or structural elucidation of secondary metabolites. The study of Xylaria cubensis resulted in the isolation of eight substances, as follows: nucleoside, diketopiperazines, isocoumarins and cytochalasins class. From Diaporthe sp. was isolated and identified eight substances, as follows: two diketopiperazines, nitropropionic acid, uracil, tyrosol, zygosporin D, pyrrolidone (unpublished) and alternariol. Colletotrichum sp. resulted in the isolation of six substances, three diketopiperazines, besides the substances N-(2-phenylethyl) acetamide, Nβ-acetyltryptamine and 3-hydroxybutan-2-yl-1H-indol-3-ylacetate (unpublished). All classes of substances produced by these endophytic fungi present several biological activities reported in the literature. Noteworthy is the phytotoxic activity of cytochalasin D against wheat coleoptile higher than commercial herbicide GOAL®. To verify the influence on the metabolic production of Xylaria cubensis, Diaporthe sp. and Colletotrichum sp., used strategies such as the co-culture on solid (PDA) and liquid medium (PDB) and epigenetic (small scale), in which endophytes showed an interesting and different metabolic production when compared with the production of monocultures and the absence of epigenetic modulator SAHA, respectively. The statistical tools, such as PCA (Principal component analysis) and PLS-DA (discriminant analysis with multivariate calibration partial least squares) allowed quick identification and location of the metabolic profiles of the co-culture compared monocultures. These data will contribute to the knowledge of the metabolic profiles, biological and enzymatic of endophytic fungi isolated from Eugenia brasiliensis and their interactions with host. / CNPq: 140980/2012-1

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.

Current energy and environmental challenges are driving the use of cellulosic materials for biofuel production. A major obstacle in this pursuit is poor ethanol tolerance among cellulolytic Clostridium species. The first objective of this work was to establish a potential upper boundary of ethanol tolerance for the cellulosome itself. The hydrolytic function of crude cellulosome extracts from C. cellulolyticum on carboxymethyl cellulose (CMC) with 0, 5, 10, 15, 20 and 25% (v/v) ethanol was determined. Results indicated that the endoglucanase activity of the cellulosome incubated in 5% and 10% ethanol was significantly different from a control without ethanol addition. Furthermore a significant difference was observed in endoglucanase activity for cellulosome incubated in 5%, 10%, 15%, 20% and 25% ethanol in a standalone experiment. Endoglucanase activity continued to be observed for up to 25% ethanol, indicating that cellulosome function in ethanol will not be an impediment to future efforts towards engineering increasing production titers to levels at least as high as the current physiological limits of the most tolerant ethanologenic microbes. The second objective of this work was to study bioethanol production by a microbial co-culture involving Clostridium cellulolyticum and a recombinant Zymomonas mobilis engineered for the utilization of oligodextrans. The recombinant Z. mobilis ZM4 pAA1 and wild type ZM4 were first tested on RM medium (ATCC 1341) containing 2% cellobiose as the carbon source. Ethanol production from the recombinant Z. mobilis was three times that observed from the wild type Z. mobilis. Concomitant with ethanol production was the reduction in OD from 2.00 to 1.580, indicating the consumption of cellobiose. No such change in OD was observed from the wild type. The recombinant ZM4 was then co-cultured with C. cellulolyticum using cellobiose and microcrystalline cellulose respectively as carbon sources. Results indicate that the recombinant ZM4 acted synergistically with C. cellulolyticum to utilize 2.0 g L-1 cellobiose, producing as much as 0.40 mM concentration of ethanol whereas only 0.20 mM ethanol was detected for the wild type ZM4 co-cultured with C. cellulolyticum under the same conditions. A co-culture of the recombinant ZM4 and C. cellulolyticum using 7.5 g L-1 microcrystalline cellulose gave lower ethanol yield than when using cellobiose. In the latter case, the recombinant began producing ethanol in 5 days whereas the wild type required 10 days to produce detectable ethanol. Future efforts will concentrate on identifying the correct concentration of cellulosic substrate at which synergy will be observed using the recombinant ZM4 and other cellulose degrading microorganisms, as well as optimizing medium formulations to better support both organisms.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20009号 / 農博第2193号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5018(農学部図書室) / 33105 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Bacteriocins are antimicrobial protein produced by certain Gram positive and negative bacteria as a defense mechanism against closely related bacteria competing for the same nutrient or in the same niche. The competition for the same nutrient is supported by the fact that bacteriocins have narrow range of effect and only likely to be effective against closely related bacteria for the same scares resources hence a bacteriocin produced by a Gram positive bacteria will be active against a Gram positive pathogens and a bacteriocin produced by a Gram negative bacteria will be active against Gram negative pathogens. This is due to the difference in cell wall composition, they are either bacteriocidal or bacteriostatic Bacteriocins have been used for thousands of years for food preservation unknowingly to man, they are considered advantageous not only to the producing bacteria, but it's now been used by the food industry as a tool to control both spoilage and pathogenic bacteria in food, in a natural manner which is acceptable to the consumer. With a lot of research been carried out on bacteriocins produced by Gram positive bacteria, antagonist to Gram positive food borne pathogens, little is known about bacteriocins produced by Gram negative bacteria which would be active against Gram negative food borne pathogens that predominate in produce. The objective of my research therefore is to screen for antimicrobial antagonist to Gram negative food borne pathogens (Escherichia coli O157:H7 and Salmonella Enteritidis) from produce, to determine an appropriate screening method, to carry out a preliminary characterization of antagonist discovered and also to determine antimicrobial spectrum of antagonist found. Lettuce was screened for antimicrobial antagonist against Gram negative pathogen (Escherichia coli O157:H7 and Salmonella Enteritidis) which were used as indicator strains With over 5000 colonies screen, 1 colony (Serratia plymuthica) was discovered to be antagonistic against these indicator strain. Further screening of cell free extract using the spot test method showed that extract from Serratia plymuthica grown alone in TSBYE showed antagonist activity against indicator strain with a little clearing on the spot of extract dropped. But extract of a co-culture of Serratia plymuthica and either Escherichia coli O157:H7 or Salmonella Enteritidis showed a more obvious clearing around spotted zone, which further indicates antagonism against indicator strains. Preliminary heat test indicates antagonist compound to be heat stable at 60oC for 30mins, 100oC for 30minutes and 60mins and 121oC for 20minites, and antagonist compound possessed antagonist activity against other strains of Escherichia coli when tested.