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Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is a fungal pathogen of maize that causes ear rots and stalk rots worldwide. In addition, it produces a group of mycotoxins called fumonisins when the fungus colonizes maize and maize-based products. Fumonisin B1 (FB1), the predominant form occurring in nature, can cause detrimental health effects in animals and humans. Several efforts were made to study the host and pathogen factors that contribute to the production of fumonisins. Using the available genomic resources, three genes with a potential role in FB1 regulation and development were identified. The genes are GBP1, GBB1 and GAP1. This research describes molecular characterization of these genes with respect to regulation of FB1 and development in F. verticillioides. GBP1 is a monomeric GTP binding protein with similarity to DRG and Obg sub-classes of G-proteins. GBB1 encodes heterotrimeric GTP binding protein β subunit. GAP1 is a GPI (Glycophosphotidylinositol) anchored protein, which belongs to a family of cell wall proteins. Targeted deletion and complementation studies indicated that GBP1 is negatively associated with FB1 biosynthesis but had no effect on conidiation in F. verticillioides. GBB1 plays an important role in regulation of FB1 biosynthesis, conidiation and hyphal growth, but not virulence. GAP1 is associated with growth, development and conidiation but not in positive regulation of FB1 or pathogenicity. The outcome of this study revealed new molecular genetic components that will help scientists better understand signal transduction pathways that regulate FB1 biosynthesis and conidiation in F. verticillioides.

Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is limited. It was previously demonstrated that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. Typically, a structural A subunit, a catalytic C subunit, and a regulatory B subunit form PP2A heterotrimer complex. Significantly, there are two PP2A regulatory subunits in F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. Based on preliminary data, I hypothesized that Ppr1 and Ppr2 are independently involved in the regulation of fumonisin biosynthesis and/or cell development, and to test this hypothesis I generated gene-deletion mutants of PPR1 and PPR2. The ppr1 deletion strain (Δppr1) resulted in drastic growth defect, but with increased microconidia production. The ppr2 deletion mutant strain (Δppr2) showed elevated fumonisin production similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cell with central septation. Δppr2 showed early hyphal branching during conidia germination. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out unique roles in regulating fumonisin biosynthesis and fungal development.

Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is a fungal pathogen of maize that causes ear rots and stalk rots worldwide. In addition, it produces a group of mycotoxins called fumonisins when the fungus colonizes maize and maize-based products. Fumonisin B1 (FB1), the predominant form occurring in nature, can cause detrimental health effects in animals and humans. Several efforts were made to study the host and pathogen factors that contribute to the production of fumonisins. Using the available genomic resources, three genes with a potential role in FB1 regulation and development were identified. The genes are GBP1, GBB1 and GAP1. This research describes molecular characterization of these genes with respect to regulation of FB1 and development in F. verticillioides. GBP1 is a monomeric GTP binding protein with similarity to DRG and Obg sub-classes of G-proteins. GBB1 encodes heterotrimeric GTP binding protein β subunit. GAP1 is a GPI (Glycophosphotidylinositol) anchored protein, which belongs to a family of cell wall proteins. Targeted deletion and complementation studies indicated that GBP1 is negatively associated with FB1 biosynthesis but had no effect on conidiation in F. verticillioides. GBB1 plays an important role in regulation of FB1 biosynthesis, conidiation and hyphal growth, but not virulence. GAP1 is associated with growth, development and conidiation but not in positive regulation of FB1 or pathogenicity. The outcome of this study revealed new molecular genetic components that will help scientists better understand signal transduction pathways that regulate FB1 biosynthesis and conidiation in F. verticillioides.

Fumonisin B1 is the most prevalent congener of the fumonisin mycotoxins produced by Fusarium verticilliodies and is considered by many to be the most toxic. Fumonisin B1 has been classified by IARC as a class 2B carcinogen. This is primarily due to evidence suggesting increased exposure to FB1 through contaminated foodstuffs is responsible for increased incidence of esophageal cancer in regions of China and South Africa. Fumonisin B1 exposure has also been implicated in the increased incidence of neural tube defects along the Texas/Mexico border. Therefore the principal goals of this research were to 1) Identify sorbent materials that would be compatible with the chemical characteristics of fumonisin B1 and evaluate their ability to sequester the toxin using established in vitro techniques; 2) evaluate urinary FB1 as a biomarker of exposure from a fumonisin contaminated diet; 3) utilize urinary FB1 as a diagnostic tool to evaluate the efficacy of NS in reducing biomarkers of FB1 bioavailability in a Ghanaian population suspected to be co-exposed to aflatoxins and fumonisins. Isothermal analysis and an alternative animal model were examined and compared to previously published results to determine the sorbent toxin interaction activity in vitro as a predictor of in vivo efficacy. An HPLC method for detection and quantitation of urinary FB1 was developed based on methods previously adapted for primary amine and biomarker analysis. Urinary FB1 was evaluated as an HPLC detectable biomarker using a rodent model. Calcium and sodium montmorillonite clays were selected to interact with the positive charge on FB1 at low pH and sorb the molecule. Ferrihydrite was selected to interact with the negative charge on the FB1 molecule at neutral to high pH. While both polarities of sorbent were effective, montmorillonite clays demonstrated a higher capacity for sorption of FB1 than ferrihydrite. These in vitro results were confirmed in a rodent model where urinary FB1 was reduced 27% in NovaSil treated rats vs. controls. Finally, in a Ghanaian population co-exposed to aflatoxins and fumonisins, urinary FB1 was significantly reduced at 2 time points when the NovaSil treatment was compared to placebo.

Synthetic efforts toward fumonisin analog were described. These are accomplished via amino acid Schiff base methodology. These efforts can be divided three major phases. First, tandem reductive alkylation with DIBAL/TRIBAL and different types of organo-lithium or Grignard nucleophiles provided threo-amino alcohol with excellent stereoselecitivites (2-27:1). The reductive alkylation utilized most hydrocarbon nucleophiles, e.g. alkyl-, vinyl-, alkenyl-, phenyl-, and dienyl-, and afforded high selectivites unless donor solvents (e.g. THF and Et2O) were used. Second, syntheses of the protected threo-γ-amino-β-hydroxy aldehydes and their stereoselectivities were introduced. The reductive alkylated threo-amino allyl alcohol was transformed via Brown’s hydroboration/oxidation protocol with 9-BBN, followed by TEMPO oxidation to give the resultant aldehydes in reasonable yields. Then, TBDPS and Schiff base protected aldehyde was coupled with phenyl- and decyl Grignard reagents to obtain predominant 3,5-anti-diols (ca. 80:20 anti:syn), characterized by ¹³C NMR analysis of Rychnovsky’s 1,3-acetonide groups. Products can be useful analogues for fumonisin and 5-hydroxy-sphingosine due to their structural similarity. Third stage involved the synthesis of C₁₁-C₂₀ fragment analog of fumonisin. Chiral auxiliaries (e.g. Evans and Myers) were administrated for stereoselective methylation, Sharpless asymmetric dihydroxylation in the presence of (DHQ)2PHAL catalyst was performed to form 1,2- syn-diols, and the manipulation of protection/deprotection and Finklestein reaction furnished C₁₁-C₂₀ fragment analog of fumonisin.

Fusarium verticillioides (Teleomorph Giberella moniliformis) is an ascomycete fungus responsible for ear and stalk rots of maize. Most importantly, it produces a group of mycotoxins called fumonisins upon colonization of maize kernels. Fumonisin B1 (FB1), the most prevalent fumonisin in nature, was first identified in 1988 and has been found to be toxic to human and animals. The gene cluster for FB1 biosynthesis and some environmental conditions responsible for the toxin production are known, but gaps in our understanding of the signaling pathways leading to FB1 biosynthesis still remain. MADS-box transcription factors (TF) are known to regulate diverse cellular functions in all eukaryotes, and in silico analyses revealed two genes, MADS1 and MAD2, in F. verticillioides. Reverse genetics studies indicated that MADS1 and MADS2 positively regulate sexual mating and FB1 biosynthesis but not pathogenicity in F. verticillioides. Furthermore, MADS1 was found to act as a broad regulator of polyketide-derived secondary metabolism. Additionally, population diversity studies were conducted in 164 F. verticillioides cultures isolated from 65 maize-producing counties in Texas. The result showed a fluid population with no particular niches formed. F. verticillioides strains were also isolated from counties that have previously tested negative for FB1 contamination in maize. The presence of the pathogen represents a risk for future FB1 contamination events if suitable conditions were to arise. My research revealed new genetic components involved in F. verticillioides secondary metabolite biosynthesis and provided a better understanding of the pathogen population fluidity in Texas.

Made available in DSpace on 2014-06-11T19:23:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-18Bitstream added on 2014-06-13T20:30:09Z : No. of bitstreams: 1 petrarca_mh_me_arafcf.pdf: 728580 bytes, checksum: fcd85cee42c96cde3425a6e4de7340fb (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / Os objetivos do presente estudo foram avaliar a influência do pH, tempo e temperatura sobre a estabilidade do derivado fumonisina B1-orto-ftaldialdeído, desenvolver e validar um método analítico para a determinação de fumonisina B1 em arroz, como também avaliar a ocorrência de fumonisina B1 em amostras de arroz polido, parboilizado polido, integral e parboilizado integral comercialiazdas no município de Araraquara, SP. Na otimização das condições cromatográficas, verificou-se que os parâmetros experimentais pH, tempo e temperatura influenciaram significativamente na estabilidade do derivado fumonisina B1-orto-ftaldialdeído. A partir de planejamento fatorial – delineamentos de Plackett & Burman e Composto Central Rotacional, e com referência no procedimento QuEChERS, foi possível deselvolver um método analítico para a determinação de fumonisina B1 em arroz. Um método de fácil execução, economicamente viável, rápido e com menor geração de resíduos orgânicos. Os parâmetros de desempenho do método analítico indicaram seletividade para a micotoxina fumonisina B1, linearidade, efeito de matriz significativo, exatidão e precisão, com limite de detecção de 50 µ g.kg-1 e limite de quantificação de 100 µg.kg-1. O método apresentou porcentagens de recuperação de 100,48, 104,97, 87,60 e 91,76% para o arroz polido, parboilizado polido, integral e parboilizado integral, respectivamente. Verificou-se contaminação por fumonisina B1 em aproximadamente 12% do total de 51 amostras analisadas. Das 20 amostras de arroz polido, apenas uma apresentou contaminação por fumonisina B1, com nível de 258,69 µ g.kg-1. Verificou-se que das 10 amostras de arroz parboilizado polido analisadas, duas apresentaram contaminação por fumonisina B1, com níveis entre 109,36 e 162,99 µg.kg-1, o que representou... / The aims of the study were to evaluate the influenc of pH, time and temperature on stability of the fumonisin B1-ortho-phthaldialdehyde derivative, develop and validate a analytical method for determination of the fumonisin B1 in rice, as also evaluate the occurrence of fumonisin B1 in polished, polished parboiled, whole and whole parboiled rice samples, marketed in Araraquara, SP. In chromatographic conditions optimizing, observed that experimental parameters pH, time and temperature significantly influenced on stability of the fumonisin B1-ortho-phthaldialdehyde derivative. From Palckett & Burman and Rotational Central Composite designs was obtained an analythical method for determination of fumonisin B1 in rice. An easy, cheap and fast method, with less generation of organic residuals. The performance parameters of the analytical method showed selectivity for the mycotoxin fumonisin B1, linearity, significant matrix effect, accuracy and precision, with detection limit of 50µg.kg-1 and limit of quantification of 100µg.kg-1. The method recovery was 100.48, 104.97, 87.60 and 91.76% for polished, polished parboiled, whole and whole parboiled rice samples, respectively. The contamination by fumonisin B1 was observed in approximately 12% of 51 samples analyzed. Contamination by fumonisin B1 was observed in approximately 12% of 51 samples analyzed. Only one polished rice sample showed contamination by fumonisin B1 whose level was of 258.69µ g.kg-1. Among the total of polished parboiled rice samples analyzed, two samples were contaminated by fumonisin B1, with levels between... (Complete abstract click electronic access below)

Orientador: Célia Maria de Sylos / Banca: Myrna Sabino / Banca: Eliane Maria Ravasi Stefano Simionato / Resumo: Os objetivos do presente estudo foram avaliar a influência do pH, tempo e temperatura sobre a estabilidade do derivado fumonisina B1-orto-ftaldialdeído, desenvolver e validar um método analítico para a determinação de fumonisina B1 em arroz, como também avaliar a ocorrência de fumonisina B1 em amostras de arroz polido, parboilizado polido, integral e parboilizado integral comercialiazdas no município de Araraquara, SP. Na otimização das condições cromatográficas, verificou-se que os parâmetros experimentais pH, tempo e temperatura influenciaram significativamente na estabilidade do derivado fumonisina B1-orto-ftaldialdeído. A partir de planejamento fatorial - delineamentos de Plackett & Burman e Composto Central Rotacional, e com referência no procedimento QuEChERS, foi possível deselvolver um método analítico para a determinação de fumonisina B1 em arroz. Um método de fácil execução, economicamente viável, rápido e com menor geração de resíduos orgânicos. Os parâmetros de desempenho do método analítico indicaram seletividade para a micotoxina fumonisina B1, linearidade, efeito de matriz significativo, exatidão e precisão, com limite de detecção de 50 µ g.kg-1 e limite de quantificação de 100 µg.kg-1. O método apresentou porcentagens de recuperação de 100,48, 104,97, 87,60 e 91,76% para o arroz polido, parboilizado polido, integral e parboilizado integral, respectivamente. Verificou-se contaminação por fumonisina B1 em aproximadamente 12% do total de 51 amostras analisadas. Das 20 amostras de arroz polido, apenas uma apresentou contaminação por fumonisina B1, com nível de 258,69 µ g.kg-1. Verificou-se que das 10 amostras de arroz parboilizado polido analisadas, duas apresentaram contaminação por fumonisina B1, com níveis entre 109,36 e 162,99 µg.kg-1, o que representou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aims of the study were to evaluate the influenc of pH, time and temperature on stability of the fumonisin B1-ortho-phthaldialdehyde derivative, develop and validate a analytical method for determination of the fumonisin B1 in rice, as also evaluate the occurrence of fumonisin B1 in polished, polished parboiled, whole and whole parboiled rice samples, marketed in Araraquara, SP. In chromatographic conditions optimizing, observed that experimental parameters pH, time and temperature significantly influenced on stability of the fumonisin B1-ortho-phthaldialdehyde derivative. From Palckett & Burman and Rotational Central Composite designs was obtained an analythical method for determination of fumonisin B1 in rice. An easy, cheap and fast method, with less generation of organic residuals. The performance parameters of the analytical method showed selectivity for the mycotoxin fumonisin B1, linearity, significant matrix effect, accuracy and precision, with detection limit of 50µg.kg-1 and limit of quantification of 100µg.kg-1. The method recovery was 100.48, 104.97, 87.60 and 91.76% for polished, polished parboiled, whole and whole parboiled rice samples, respectively. The contamination by fumonisin B1 was observed in approximately 12% of 51 samples analyzed. Contamination by fumonisin B1 was observed in approximately 12% of 51 samples analyzed. Only one polished rice sample showed contamination by fumonisin B1 whose level was of 258.69µ g.kg-1. Among the total of polished parboiled rice samples analyzed, two samples were contaminated by fumonisin B1, with levels between... (Complete abstract click electronic access below) / Mestre