N-acylethanolamines (NAEs) with C12-C18 acyl chain are ubiquitous in seed plants and play a role in mediating abscisic acid (ABA)-dependent or -independent responses to stress. In moss Physcomitrella patens, using selective lipidomics approach, we recently identified the occurrence of anandamide or N-arachidonylethanolamide (NAE 20:4) and its precursors that were previously not reported in plants. Occurrence of anandamide in moss provides us with a unique opportunity to address if early land plants retained NAE-mediated signaling mechanism that is akin to animals but not to vascular plants. It is hypothesized that a distinctive NAE profile and metabolic pathway occurs in P. patens. To this extent, putative genes that might be responsible for anandamide metabolic pathway were identified and their expression levels were determined for three developmental stages of moss. The NAE metabolite levels and transcript levels for putative genes were higher in protonema stage and anandamide showed higher growth inhibitory effects, chlorophyll reduction, and putative gene induction than NAE 12:0, compared to ABA, when applied exogenously.

Colorectal cancer is the 4th most widespread cause of cancer mortality. One of the pathways that are involved in the development of colorectal cancer is the arachidonic acid metabolizing lipoxygenase (LOX) pathway. Inflammatory molecules formed from this pathway exert profound effects that may exacerbate the development and progression of colon and other cancers. 15 lipoxygenase-1 (15-LOX-1) is a member of LOX protein family that metabolizes primarily linoleic acid to 13-(S)-HODE. Several lines of evidence support an antiangiogenic role for 15-LOX-1, especially through 13-(S)-HODE. The expression of 15-LOX-1 is lost in colon cancer cells. Our aim in this thesis was to study whether 15-LOX-1 expression has an anticarcinogenic role, particularly on the metastatic and angiogenic potential of colon cancer cells. For this purpose, 15-LOX-1 was introduced into HCT-116 colon cancer cell lines. Having confirmed 15-LOX-1 expression and activity it was observed that expression of 15-LOX-1 significantly decreased cell proliferation, cell motility, anchorage-independent growth, migration and invasion across Matrigel, the expression of the metastasis-related MTA-1 protein, neoangiogenesis and induced apoptosis. Mechanistically, most of these effects were arbitrated by the 15-LOX-1 mediated inhibition of the inflammatory transcription factor NF-&kappa / B via the orphan nuclear receptor PPAR&gamma / . In conclusion, we propose that 15-LOX-1 has anti-tumorigenic properties and can be exploited for therapeutic benefits.

Colorectal cancer is the 4th most widespread cause of cancer mortality. One of the pathways that are involved in the development of colorectal cancer is the arachidonic acid metabolizing lipoxygenase (LOX) pathway. Inflammatory molecules formed from this pathway exert profound effects that may exacerbate the development and progression of colon and other cancers. 15 lipoxygenase-1 (15-LOX-1) is a member of LOX protein family that metabolizes primarily linoleic acid to 13-(S)-HODE. Several lines of evidence support an antiangiogenic role for 15-LOX-1, especially through 13-(S)-HODE. The expression of 15-LOX-1 is lost in colon cancer cells. Our aim in this thesis was to study whether 15-LOX-1 expression has an anticarcinogenic role, particularly on the metastatic and angiogenic potential of colon cancer cells. For this purpose, 15-LOX-1 was introduced into HCT-116 colon cancer cell lines. Having confirmed 15-LOX-1 expression and activity it was observed that expression of 15-LOX-1 significantly decreased cell proliferation, cell motility, anchorage-independent growth, migration and invasion across Matrigel, the expression of the metastasis-related MTA-1 protein, neoangiogenesis and induced apoptosis. Mechanistically, most of these effects were arbitrated by the 15-LOX-1 mediated inhibition of the inflammatory transcription factor NF-&kappa / B via the orphan nuclear receptor PPAR&gamma / . In conclusion, we propose that 15-LOX-1 has anti-tumorigenic properties and can be exploited for therapeutic benefits.

Air pollutant exposure poses a health risk to humans and impacts negatively on agriculture. High levels of air pollution resulted in extensive crop damage and yield reduction in Europe and USA. The Highveld region in South Africa, a very important area for maize and soya production, has already been declared an air pollution hot spot, with SO2 being the most concerning air pollutant. Most of the SO2 over the Highveld originates from the burning of coal for power generation. Developing countries, such as South Africa, are highly dependent on agriculture for food security and high levels of air pollution pose serious risks to the agricultural industry. Currently very little information is available on the effects of air pollution on crop production in South Africa. This study aimed to establish exposure-response relationship for SO2 on soybean and the quantification thereof on the morphological, physiological and biochemical characteristics. Two soybean cultivars were used, namely: LS 6164 and PAN 1666. The plants were fumigated for 7 hours, 7 days a week with 0 (carbon filtered control; CF), 25, 75 and 150 ppb SO2. The effect of SO2 was investigated on the growth, photosynthetic capabilities, photosynthetic gas exchange, peroxidase activity and lipoxygenase activity of the cultivars. Foliar injuries and interveinal chlorosis were visible with increasing levels of SO2 as well as a decrease in biomass accumulation, especially in root biomass; a more prominent feature of LS 6164. The number of nodules of both cultivars decreased insignificantly as the levels of SO2 increased. The number of pods per plant and the average weight of 30 seeds indicated a downward trend with an increase in SO2 concentration. The chlorophyll content of PAN 1666 was lower compared to LS 6164. PAN 1666 had the largest reduction in stomatal conductance at 150 ppb SO2 fumigation. The photosynthetic vitality index indicated that LS 6164 was more sensitive to SO2 inhibition from 25 ppb SO2 and higher, whereas PAN 1666 mostly became sensitive to SO2 from 75 ppb SO2. A decrease in the ability to absorb light energy, the trapping of excitation energy to transfer electrons beyond QA-, and the reduction of end electron acceptors all contributed to the decline in the vitality index. Sulphur content increased significantly in the 75 ppb and 150 ppb treatments of both cultivars. Induced peroxidase and lipoxygenase activity was seen in both cultivars, especially at higher concentrations of SO2 treatments. PAN 1666 had a higher rate of peroxidase and lipoxygenase activity compared to LS 6164. The implication for SO2 on crop production in the highly industrial Highveld area was demonstrated to be potentially of great concern. The dose-response relationships plotted for OJIP parameters emphasized that SO2 is an inhibitor of photosynthesis and phytotoxic of nature. Both cultivars experienced limitations from 75 ppb, especially at the 150 ppb SO2 concentration. From these results it appears that PAN 1666 is more adapted to SO2 compared to LS 6164 and levels of 75 ppb SO2 and higher become toxic to these plants. / Thesis (Master of Environmental Sciences)--North-West University, Potchefstroom Campus, 2013

Air pollutant exposure poses a health risk to humans and impacts negatively on agriculture. High levels of air pollution resulted in extensive crop damage and yield reduction in Europe and USA. The Highveld region in South Africa, a very important area for maize and soya production, has already been declared an air pollution hot spot, with SO2 being the most concerning air pollutant. Most of the SO2 over the Highveld originates from the burning of coal for power generation. Developing countries, such as South Africa, are highly dependent on agriculture for food security and high levels of air pollution pose serious risks to the agricultural industry. Currently very little information is available on the effects of air pollution on crop production in South Africa. This study aimed to establish exposure-response relationship for SO2 on soybean and the quantification thereof on the morphological, physiological and biochemical characteristics. Two soybean cultivars were used, namely: LS 6164 and PAN 1666. The plants were fumigated for 7 hours, 7 days a week with 0 (carbon filtered control; CF), 25, 75 and 150 ppb SO2. The effect of SO2 was investigated on the growth, photosynthetic capabilities, photosynthetic gas exchange, peroxidase activity and lipoxygenase activity of the cultivars. Foliar injuries and interveinal chlorosis were visible with increasing levels of SO2 as well as a decrease in biomass accumulation, especially in root biomass; a more prominent feature of LS 6164. The number of nodules of both cultivars decreased insignificantly as the levels of SO2 increased. The number of pods per plant and the average weight of 30 seeds indicated a downward trend with an increase in SO2 concentration. The chlorophyll content of PAN 1666 was lower compared to LS 6164. PAN 1666 had the largest reduction in stomatal conductance at 150 ppb SO2 fumigation. The photosynthetic vitality index indicated that LS 6164 was more sensitive to SO2 inhibition from 25 ppb SO2 and higher, whereas PAN 1666 mostly became sensitive to SO2 from 75 ppb SO2. A decrease in the ability to absorb light energy, the trapping of excitation energy to transfer electrons beyond QA-, and the reduction of end electron acceptors all contributed to the decline in the vitality index. Sulphur content increased significantly in the 75 ppb and 150 ppb treatments of both cultivars. Induced peroxidase and lipoxygenase activity was seen in both cultivars, especially at higher concentrations of SO2 treatments. PAN 1666 had a higher rate of peroxidase and lipoxygenase activity compared to LS 6164. The implication for SO2 on crop production in the highly industrial Highveld area was demonstrated to be potentially of great concern. The dose-response relationships plotted for OJIP parameters emphasized that SO2 is an inhibitor of photosynthesis and phytotoxic of nature. Both cultivars experienced limitations from 75 ppb, especially at the 150 ppb SO2 concentration. From these results it appears that PAN 1666 is more adapted to SO2 compared to LS 6164 and levels of 75 ppb SO2 and higher become toxic to these plants. / Thesis (Master of Environmental Sciences)--North-West University, Potchefstroom Campus, 2013

In this study, the efficiency of high-pressure treatment (HHP, 250 - 450 MPa) with the combination of heat treatment (20 - 70oC) on peroxidase (POD) and lipoxygenase (LOX) inactivation in green beans, green peas and carrots was investigated for blanching purposes. Two steps treatments were also performed by pressurization at 250 MPa at 20&deg / C for 15- 60 min and then by water blanching at 40-70&deg / C. For green beans, 25 % residual activity was obtained by water blanching at 50&deg / C for 15 min after the pressurization at 250 MPa for 60 min. The enzyme inactivation in green peas was 78 % with water blanching at 50oC for 30 min after holding at 250 MPa for 60 min. When the carrots were water blanched at 50&deg / C for 30 min after HHP treatment at 250 MPa for 15min, 13 % residual activity was obtained. During the experiments, the stability gain or the activation of latent form of the enzymes were observed prior to inactivation. For carrots, LOX activity could not be measured. For green beans, 22 % LOX inactivation was obtained by holding at 250 MPa for 15 min and then by water blanching at 40&deg / C for 5 min. For green peas, the multiple treatment of 250 MPa for 30 min and water blanching at 50&deg / C for 30 min provided 70% inactivation. To obtain the enzyme inactivation higher than 90 % for blanching purposes, the pressure applied must be increased. Key words: high hydrostatic pressure, green bean, green pea, carrot, blanching, peroxidase, lipoxygenase

Il Fusarium verticillioides (Fv) causa il marciume rosa della spiga e contamina le cariossidi con fumonisine, una famiglia di micotossine che colpisce mangimi e alimenti considerata cancerogena per l'uomo e gli animali. Sono stati condotti diversi studi per identificare i geni del mais associati alla resistenza della pianta ospite all'infezione da Fv e l'accumulo di fumonisina. È noto che le ossilipine regolano la difesa contro i patogeni e che il cross-talk lipidico ospite-patogeno influenza la patogenesi. A questo proposito, i mutanti di mais trasposonici del gene ZmLOX4, la linea suscettibile W22 e la resistente TZI18 sono stati testati per la resistenza a Fv mediante il saggio biologico Rolled Towel Assay (RTA). Inoltre, sono stati studiati i profili di espressione di 16 geni coinvolti nella via LOX e volatili verdi (GLV) e l'attività della lipossigenasi è stata analizzata nelle stesse linee. Inoltre, è stata applicata la tecnologia di modifica del genoma di Clustered Shortspeed Palindromic Repeat / Cas9 associato (CRISPR / Cas9) regolarmente esaminata per indagare le possibili implicazioni del gene ZmLOX6 e del fattore di trascrizione ZmWRKY125 nei meccanismi di resistenza contro Fv. L'espressione di questi geni è stata precedentemente osservata dagli esperimenti di RNA - Seq in genotipi resistenti al mais e Studi di Genome Wide Association (GWAS) che hanno portato a un SNP significativamente associato a ZmWRKY125. Inoltre, il gene ZmLOX4 è stato overespresso nella linea A188 per valutare un possibile miglioramento della resistenza alla malattia verso Fv. Il lavoro molecolare del CRISPR si basa su una doppia clonazione utilizzando due diverse single guide RNA (sgRNA) per un bersaglio genico. I costrutti sotto il promotore ZmpUBI nel vettore binario p1609 sono stati trasformati nella linea A188 utilizzando la trasformazione mediata da Agrobacterium tumefaciens. Le piante di mais modificate nei geni ZmLOX6 e ZmWRKY125 e ZmLOX4 che sovraesprimono saranno caratterizzate per RTA, prove sperimentali in campo e per il loro contenuto di fumonisina. Inoltre, saranno testati l’attività lipossigenasica totale, i suoi metaboliti derivati ​​e le osslipine, oltre all'analisi dell'espressione dei principali geni coinvolti nella via dell'acido jasmonico. / Fusarium verticillioides (Fv) causes ear rot in maize and contaminates the kernels with fumonisins, a family of mycotoxins that affects feed and food and considered carcinogenic for humans and animals. Several studies were conducted to identify maize genes associated with host plant resistance to Fv infection and fumonisin accumulation. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defense against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. In this regard, maize mutants carrying Mu insertions in the ZmLOX4 gene, the susceptible W22 and the resistant TZI18 lines were tested for Fv resistance by the screening method rolled towel assay (RTA). Additionally, the expression profiles of 16 genes involved in the LOX and green leaves volatiles (GLV) pathway were studied and the lipoxygenase activity was investigated in the same lines as well. Furthermore, the genome editing technology of Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9) was applied in order to investigate the possible implication of the lipoxygenase gene ZmLOX6 and the transcription factor ZmWRKY125 in the resistance mechanisms against Fv. The enhanced expression of these genes was previously observed by RNA - Seq experiments in maize resistant genotypes and Genome Wide Association Studies (GWAS) resulted in one SNP significantly associated with ZmWRKY125. Moreover, the gene ZmLOX4 was over-expressed in the line A188 for evaluating a possible improvement of the disease resistance towards Fv. The CRISPR cloning was based on a double cloning using two different guides (sgRNA) for one gene target. The constructs under the maize promoter ZmpUBI in the binary vector p1609 were transformed into the maize A188 line using Agrobacterium tumefaciens mediated transformation. Maize plants edited in the genes ZmLOX6 and ZmWRKY125, and over-expressing ZmLOX4 will be characterized for Fv resistance using rolled towel assay, field assay and for their fumonisin content. Furthermore, the content of jasmonic acid, its derivative metabolites, and oxylipins will be tested, as well as the expression analysis of the main genes involved in the jasmonic acid pathway will be performed.

Muitas substâncias das espécies de Lychnophorinae (Asteraceae) são relatadas como inibidoras da síntese de mediadores da cascata do processo inflamatório. Nesse processo, duas enzimas são essenciais e atuam no metabolismo do ácido araquidônico, formado em processos inflamatórios: ciclooxigenase (COX) e lipoxigenase (LOX). A análise de impressões digitais metabólicas é um método capaz de fornecer informações sobre o objeto de estudo através da utilização de ferramentas estatísticas, além de possibilitar a correlação desses dados com outros utilizando métodos in silico. O objetivo deste estudo foi analisar 26 espécies da subtribo Lychnophorinae quanto à atividade inibitória in vitro das vias COX- e LOX-dependentes e identificar as substâncias responsáveis por essa atividade através de análises in silico de correlação entre bioatividade e impressão digital metabólica. As impressões digitais metabólicas dos extratos hidrometanólicos das espécies de Lychnophorinae foram obtidas utilizando UHPLC-UV-(DAD)-MS (OrbitrapTM). Os ensaios de triagem de inibição das vias COX-1 e 5-LOX-dependentes revelaram que 20 espécies possuem atividade inibitória dupla para ambas as vias com valores de IC50 menores que 100 ?g.mL-1. Dentre essas, 11 espécies apresentaram valores de IC50 menores que 40 ?g.mL-1, e cinco apresentaram valores de IC50 menores que 10 ?g.mL-1. Utilizando-se as impressões digitais metabólicas e os resultados de inibição enzimática foram realizadas análises estatísticas multivariadas supervisionadas e não supervisionadas (PCA, PLS e OPLS) visando à identificação de substâncias discriminantes (ativas). Através das análises de correlação, obtiveram-se as prováveis substâncias que detém o potencial inibitório. As cinco substâncias com maior potencial discriminante foram identificadas por técnicas de desreplicação e são: a lactona sesquiterpênica (4,5-diidro-15-desoxigoyazensolido), dois flavonóides glicosilados (3-O-(acetil-hexosídeo)-quercetina e 7-O-(cumaroil-hexosídeo)-apigenina), e um hidroxinerolidol. Assim, este estudo revelou o ótimo potencial inibidor das enzimas COX-1 e 5-LOX dos extratos hidrometanólicos das espécies de Lychnophorinae. Além disso, indicou as substâncias mais discriminantes responsáveis por essa atividade, sendo as substâncias acima mencionadas as propostas como as principais responsáveis pela atividade inibidora das enzimas COX e LOX / Several compounds from Lychnophorinae species (Asteraceae) are reported as inhibitors of cascade mediators that elicits the inflammatory process. During this process, two enzymes are essential in the metabolism of the arachidonic acid: cyclooxygenase (COX) and lipoxygenase (LOX). The analysis of metabolic fingerprinting is a method that provides information about the object of study by using statistical tools, and enables the correlation of these data with others using in silico methods. This work therefore aimed to analyze 26 species of the Lychnophorinae subtribe for in vitro inhibition of COX-1 and 5-LOX and (to) identify the bioactive compounds responsible for this activity by in silico correlation analysis between bioactivity and metabolic fingerprint. The metabolomic analysis was carried out using UHPLC-DAD-ESI-Orbitrap and a metabolic fingerprint for each extract was obtained. The in vitro inhibition screening assays of COX-1 and 5-LOX revealed that 20 extracts presented dual inhibitory activity on both enzymes with IC50 values lower than 100 ?g.mL-1. Among them, 11 species showed IC50 values lower than 40 ?g.mL-1 and five lower than 10 ?g.mL-1. In order to identify discriminant substances (active), supervised and non-supervised multivariate statistical analysis (PCA, PLS e OPLS) were performed using the metabolic fingerprints and the results of enzyme inhibition. Through correlation analysis, it was possible to locate the substances most likely to be responsible for the pharmacological activity in both enzymes simultaneously; among them, five were chosen as the most likely. The substances were identified by dereplication as: a sesquiterpene lactone (4,5-dihydro-15-desoxygoyazensolide), two flavonoids (3-O-(acetil-hexoside)-quercetin and 7-O-(cumaroil-hexoside)-apigenine), and a hidroxynerolidol. In summary, in this work it was possible to reveal crude extracts with outstanding inhibitory potential of both, COX-1 and 5-LOX, enzymes as well as to propose the most probable compounds responsible for this action, and the compounds mentioned above were proposed as the main responsible for the inhibitory activity.

no / Lipid mediators are produced from the oxidation of polyunsaturated fatty acids through enzymatic and free radical-mediated reactions. When subject to oxygenation via cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases, polyunsaturated fatty acids give rise to an array of metabolites including eicosanoids, docosanoids, and octadecanoids. These potent bioactive lipids are involved in many biochemical and signaling pathways, with inflammation being of particular importance. Moreover, because they are produced by more than one pathway and substrate, and are present in a variety of biological milieus, their analysis is not always possible with conventional assays. Liquid chromatography coupled to electrospray mass spectrometry offers a versatile and sensitive approach for the analysis of bioactive lipids, allowing specific and accurate quantitation of multiple species present in the same sample. Here we explain the principles of this approach to mediator lipidomics and present detailed protocols for the assay of enzymatically produced oxygenated metabolites of polyunsaturated fatty acids that can be tailored to answer biological questions or facilitate assessment of nutritional and pharmacological interventions.

No description available.