Steroid and eicosanoid metabolism occurs in two phases and primarily within hepatic tissues, but localized metabolism has been examined in several extra-hepatic tissues in humans and rodents. Phase I of metabolism is performed by Cytochrome P450s (CYP) that add hydroxyl groups to the carbon ring structure which is further metabolized by phase II UDP-glucuronosyltransferase (UGT). The overall objectives of the following experiments were to: 1) determine the amount of extra-hepatic steroid metabolism within reproductive tissues of cattle across the estrous cycle; 2) determine the amount of extra-hepatic steroid metabolism and an oxylipin profile within reproductive tissues of cattle based on pregnancy status; and 3) determine the amount of endometrial blood perfusion in cattle using a novel laser Doppler technique. Activity of CYP1A was found within corpora lutea (CL) tissues of both pregnant and non-pregnant cattle, but not within endometrial tissues. Endometrial perfusion, measured using a novel laser Doppler technique, was also validated by measuring angiogenic factors in close proximity to the location of perfusion. A positive correlation (r = 0.28; P = 0.04) was observed between endometrial perfusion and nitrite concentration, an angiogenic factor. Endometrial blood perfusion was affected by the proximity to the CL, but not by the proximity of the dominant follicle. In addition, UGT was categorized across the estrous cycle and the activity was dependent upon the proximity of the CL. Oxylipins, including eicosanoids, were also profiled in CL of cattle that were non-pregnant and pregnant with 5 out of 39 oxylipins differentially expressed. The activity and oxylipin products of steroid and eicosanoid enzymes were not correlated with serum or luteal progesterone. Through these experiments, we have verified that there is localized metabolism of steroids and eicosanoids within reproductive tissues of cattle as well as fetal tissues. Also, we have achieved a full oxylipin profile of non-pregnant and pregnant cattle CL with five oxylipins contained in various amounts between pregnancy status.

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defense responses to pathogens and pests. In contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. We cloned and characterized three maize LOXs ZmLOX10 ZmLOX11 and ZmLOX12. Both ZmLOX10 and ZmLOX11 apeared to be 13-LOX, whereas ZmLOX12 is a unique 9-LOX. Whereas leaf was the preferential site of ZmLOX10 expression, ZmLOX11 was strongly expressed in silks. Induction of these ZmLOX10 and ZmLOX12 by wounding and defense-related compounds suggested their role in plant resistance mechanisms against pests and pathogens. Abscisic acid, however, was the only inducer of ZmLOX11 in leaves. Higher increase in ZmLOX10 transcripts in maize infected by fungus Cochliobolus carbonum implicated this gene in resistance responses to necrotrophic pathogens. In addition, ZmLOX10 was shown to be the first reported LOX to be regulated by a circadian clock. It was found that ZmLOX10 was also inducible by low temperatures. Phenotypical studies of wild type and mutant near isogenic lines showed that expression of ZmLOX12, specific to underground organs, was required for pathogenesis of F. verticillioides on maize mesocotyls.

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defense responses to pathogens and pests. In contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. We cloned and characterized three maize LOXs ZmLOX10 ZmLOX11 and ZmLOX12. Both ZmLOX10 and ZmLOX11 apeared to be 13-LOX, whereas ZmLOX12 is a unique 9-LOX. Whereas leaf was the preferential site of ZmLOX10 expression, ZmLOX11 was strongly expressed in silks. Induction of these ZmLOX10 and ZmLOX12 by wounding and defense-related compounds suggested their role in plant resistance mechanisms against pests and pathogens. Abscisic acid, however, was the only inducer of ZmLOX11 in leaves. Higher increase in ZmLOX10 transcripts in maize infected by fungus Cochliobolus carbonum implicated this gene in resistance responses to necrotrophic pathogens. In addition, ZmLOX10 was shown to be the first reported LOX to be regulated by a circadian clock. It was found that ZmLOX10 was also inducible by low temperatures. Phenotypical studies of wild type and mutant near isogenic lines showed that expression of ZmLOX12, specific to underground organs, was required for pathogenesis of F. verticillioides on maize mesocotyls.

Total synthesis of natural products is of contemporary interest in organic synthesis. One of the useful ways to synthesize the natural products is to originate from inexpensive chiral pool compounds abundantly available in nature. In this context, our research group is actively involved in the use of tartaric acid as the four carbon four hydroxy building block in the synthesis of a number of natural products of therapeutic importance. Our strategy relies on the utility of γ-hydroxy amides derived from tartaric acid involving a controlled addition of Grignard reagents and stereoselective reduction. We were successful in application o f this useful building block for the synthesis of a variety of natural products possessing varied functional groups (Chart-1). derived from tartaric acid in the synthesis of oxylipins such as pinellic acid and diyne containing natural products. Chapter 1 of the thesis describes the total synthesis of (+) pinellic acid 6 and (Z,8S,9S,10R)-8,9,10-trihydroxyoctadec-6-enoic acid 10. Key strategy in the synthesis of pinellic acid is elaboration of the aldehyde 3, derived from the γ-hydroxy amide 2 via Horner-Emmons-Wadsworth reaction to yield the α,β-unsaturated ketone 4. Stereoselective reduction of the ketone with (R)-BINAL-H produced the alcohol with requisite stereochemistry which was further extended to pinellic acid 6 (Scheme 1). Wittig homologation of the aldehyde 8 derived from γ-hydroxy amide 7 is the key step for the synthesis of the (Z,8S,9S,10R)-8,9,10-trihydroxyoctadec-6-enoic acid 10. Second chapter of the thesis deals with total synthesis of diyne containing natural products. In the first part of this chapter enantioselective synthesis of possible diastereomers of heptadeca1-ene-4,6-diyne-3,8,9,10-tetrol, a structure proposed for the natural product isolated from Hydrocotyle leucocephala, is accomplished. The alkyne precursors 13 and 14 were synthesized from the α-hydroxy ester 12 derived from γ-hydroxy amide 11 while the alkyne 17 is synthesized from the masked tetrol 16 derived from lactol 15 which was obtained from D-ribose. yne to assemble the diyne unit which was further elaborated to heptadeca-1-ene-4,6-diyne3,8,9,10-tetrol (Scheme 3). It was found that the NMR spectral data of the putative structure assigned for the natural product did not match with any of the diasteromers that were synthesized. This establishes that the structure proposed for the natural product is wrong and requires revision. OH OH OH 18 OH OH 19 OH OH 20 OH OH Scheme 3: Synthesis of diastereomers of heptadeca-1-ene-4,6-diyne-3,8,9,10-tetrol. [Part of this work is published: Prasad, K. R.; Swain, B. J. Org. Chem. (in press)] Second part of this chapter deals with the synthesis of panaxytriol 26 and panaxydiol 28. Key reaction in the synthesis of panaxytriol and panaxdiol is the coupling of bromoalkynes 25 and 27 with 3-silyloxy pent-1-en-4-yne and further elaboration to the triol and diol. The required alkynes were synthesized from the primary alcohol 24 which was obtained from the γ-hydroxy amide 11 involving a series of simple synthetic operations. (Scheme 4). (For structural formula pl see the abstract file)

No description available.

Les champignons endophytes sont des microorganismes non-pathogènes impliqués dans des associations mutualistes avec les plantes. Les endophytes foliaires, en particulier, représentent un groupe très divers, mais leurs interactions avec la plante hôte et ses micro-organismes associés sont peu connues. Des travaux préliminaires initiés par notre équipe, explorant la diversité microbienne foliaire du conifère Cephalotaxus harringtonia, ont permis d’isoler la souche fongique Paraconiothyrium variabile (Ascomycota), un antagoniste du phytopathogène Fusarium oxysporum. Au cours de leur interaction, on détecte des quantités moindres de beauvéricine, une mycotoxine synthétisée par F. oxysporum. En parallèle, on observe une augmentation de la synthèse de deux oxylipines, l’acide 13-hydroperoxyoctadécadiénoïque (13-HPODE) et l’acide 13-oxo-octadécadiénoïque (13-oxo-ODE), dans la zone de confrontation. L'objectif de ce travail était de comprendre les mécanismes conduisant à la diminution de la beauvéricine au cours de l'interaction et d'explorer le rôle des oxylipines dans la régulation de cette dernière. Dans mon travail de thèse, je montre la présence de deux gènes lox chez P. variabile (pvlox1 et pvlox2) codant tous deux pour des manganèse lipoxygénases, potentiellement à l'origine des acides 13-HPODE et 13-oxo-ODE. Pvlox2 est spécifiquement induit pendant l'interaction, et ces résultats sont corroborés par une synthèse accrue de 13-HPODE chez P. variabile. Par ailleurs, l'interaction avec l’endophyte, ainsi que l'ajout de l’oxylipine 13-HPODE, régulent positivement la voie de biosynthèse de la beauvéricine, comme l’indiquent les teneurs plus élevées en mycotoxines observées chez F. oxysporum. Enfin, nous avons montré que la beauvéricine inhibait la croissance de l’endophyte, mais que ce dernier était capable de dégrader la mycotoxine, expliquant ainsi les faibles quantités de beauvericine observées initialement dans la zone de compétition. Ce travail contribue à la compréhension du rôle des oxylipines dans la communication inter-microbienne. / Endophytic fungi are non-pathogenic microorganisms involved in mutualistic associations with their host. Foliar endophytes, in particular, represent a very diverse group but little is known about their interactions with the host and its associated micro-organisms. In preliminary work, exploring the leaf microbial diversity of the conifer Cephalotaxus harringtonia, our team isolated the fungal strain Paraconiothyrium variabile (Ascomycota), an antagonist of the phytopathogen Fusarium oxysporum. During their interaction, decreased amounts of the F. oxysporum mycotoxin beauvericin, and higher amounts of the two oxylipins, 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-oxo-octadecadienoic acid (13-oxo-ODE), were observed in the confrontation zone. The objective of the present work was to understand the mechanisms leading to beauvericin decrease during the interaction and to explore the role of oxylipins in beauvericin regulation. In my thesis work I show the presence of two lox genes in P. variabile (pvlox1 and pvlox2) coding both for manganese lipoxygenases, potentially at the origin of 13-HPODE and 13-oxo-ODE. Pvlox2 is specifically induced during the interaction, which lead to an increased synthesis of 13-HPODE in P. variabile. The endophyte itself, as well as the oxylipin 13-HPODE, up-regulated the beauvericin biosynthesis gene beas, which was paralleled by higher mycotoxin content in the mycelium of F. oxysporum. Finally, we showed that beauvericin inhibited the endophyte’s growth, but the latter was capable to degrade the mycotoxin, which explains the lower amounts of beauvericin found in the competition zone. This work presents pioneer undertaking to elucidate the role of oxylipins in inter-microbial crosstalk.

Lipoxygenase (LOX)-derived oxylipins are known to play critical roles in defense against herbivores and pathogens. The objective of this study was to determine the biochemical, molecular and physiological roles of a specific maize lipoxygenase gene, ZmLOX10, with special emphasis on LOX10-derived oxylipins in plant-insect and plant-pathogen interactions. To achieve this goal, independent mutant alleles were generated and genetically advanced to create near-isogenic mutant and wild-type lines suitable for functional analysis. Here we provide genetic evidence that LOX10 is the sole LOX isoform in maize required for the biosynthesis of green leafy volatiles (GLV) in leaves and show that LOX10- mediated GLVs play a significant role in direct and indirect defense responses to insects through their regulation of jasmonic acid and volatile organic compound production. Contrary to the defensive role of LOX10 in plant-insect interactions, tests for susceptibility to fungal pathogens suggest that LOX10-mediated GLVs may contribute to the development of disease symptoms to the economically important maize pathogens, Aspergillus flavus and Colletotrichum graminicola. Specifically, LOX10-derived GLVs may facilitate aflatoxin accumulation in response to A. flavus infection and may play a positive role in anthracnose leaf blight and stalk rot caused by C. graminicola. Collectively, our results suggest that metabolites derived from GLV-regulated pathways have a significant impact on molecular plant-herbivore and plant-pathogen interactions.

Allergic airway disease, i.e. allergic rhinitis (AR) and asthma, is a common health problem. The prevalence is increasing in most countries of the world. Traffic-related air pollution has been found to induce and enhance allergic airway disease, but the underlying mechanisms are not known. Oxylipins are fatty acid metabolites, of which several have been linked to asthmatic airway inflammation. Oxylipin profiles have previously been investigated in bronchoalveolar lavage (BAL), mainly reflecting the peripheral lung, but not in bronchial wash (BW), which better reflect the proximal airways. The airway epithelium is covered by a respiratory tract lining fluid (RTLF) The RTLF contains antioxidants to protect from oxidative stress, which may be caused by exposure to air pollution. Previous studies have reported diminished levels of the antioxidant ascorbate (vitamin C) in the RTLF of patients with asthma. Little is known about the regulation of vitamin C in the lung. The aim of this thesis was to investigate airway inflammatory responses to diesel exhaust exposure in patients with AR and allergic asthma; to evaluate oxylipin profiles in different regions of the lung in patients with allergic asthma; and to study the distribution of vitamin C transporters in the airways of patients with allergic asthma. Diesel exhaust (PM10 100 μg/m3 for 2 h) induced a neutrophilic airway inflammation in healthy individuals evaluated 18 h after exposure. Patients with AR and asthma did not respond with an enhanced airway inflammation. However, a small increase in myeloperoxidase was found in BAL from patients with AR, as well as decreases in epithelial tryptase and BW stem cell factor. This indicates that other mechanisms than classical inflammation are responsible for the increased sensitivity to traffic-related air pollution in patients with allergic airway disease. Oxylipin baseline profiles differed between peripheral and proximal airways in both allergic asthmatics and healthy individuals. Total oxylipin concentrations, and five individual oxylipins, primarily from the lipoxygenase (LOX) pathway, were elevated in BW from asthmatics compared to healthy controls, supported by immunohistochemical staining of 15-LOX-1 in the bronchial epithelium. This suggests that lung compartment-specific sampling should be considered in future studies. Sodium dependent vitamin C transporter 2 (SVCT2) was, for the first time, found present in the human lung epithelium, localised mainly within goblet cells. A negative correlation between SVCT2+ goblet cells and vitamin C suggests that these cells may play a hitherto unknown function in ascorbate re-uptake and recycling at the air-lung interface.

Flocculation, the process whereby yeast cells attach in groups and sediment to the top or bottom of a fermenter, is industrially important in many fermentation batch operations. These batch operations include wine, distilled spirits, cider, bio-ethanol and production of commercial yeast metabolites. In the case of brewing yeast, it has been determined that flocculation occurs due to three forces called hydrophobic interactions, zymolectin binding and to a lesser extent, surface charge neutralization. This project sought to more closely study hydrophobic interactions and zymolectin binding. Earlier studies had shown that certain hydrophobic carboxylic acids, 3-OH oxylipins, formed in brewing yeast at flocculation onset. Therefore, these compounds showed potential as an indicator of overall cell surface hydrophobicity, and it was believed that flocculation level, cell surface hydrophobicity and oxylipin level would increase in unison in the yeast cell during brewing fermentations. During lab scale fermentations in shaker flasks and in a miniature fermentation assay setup, both flocculation level and cell surface hydrophobicity increased coincidently. However, 3-OH oxylipins could not be isolated from whole cells or cell wall isolates grown in the shaker flasks or whole cells grown in the miniature fermentation assay at detection limits approximated as 50 ng/0.5 g wet yeast. Due to their minute levels in brewing yeast cells, it was proposed that 3-OH oxylipins may mediate flocculation and aggregation via a quorum sensing mechanism instead of by increasing cell surface hydrophobicity. A disagreement exists in the literature where certain researchers believe zymolectin activity is induced, while others believe it is constitutive. The second part of this study attempted to address this by measuring zymolectin density during lab scale fermentations with a flow cytometer. Because of flow cytometry’s capacity for multiparametric analysis, large amounts of data were produced which gave information on not only zymolectin density, but also cell size and cellular complexity. Upon statistical analysis of the data, it was not possible to either refute or confirm the claim that zymolectin activity is induced or constitutive. However, the results suggested there could have been a population of cells with fewer zymolectins, and this certainly warrants further investigation. During the lab scale fermentations, cell size measured by a flow cytometer appeared to be correlated with manual measures of cell size. Furthermore, cell size tended towards uniformity during the fermentation which has also been observed in similar studies employing flow cytometry. Conversely, the cellular complexity of the yeast in this study did not change as in other studies by this may have been due either to strain differences or the methods used herein.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 5 uppsatser.