There is a wealth of scientific evidence associating the dietary intake of omega 3 long-chain polyunsaturated fatty acids with beneficial health properties. In this study, alternative natural sources of these polyunsaturated fatty acids are sought from novel low temperature isolated fungi. Over 100 low temperature isolated fungi were screened for very long chain polyunsaturated fatty acids (VLCPUF As), such as C20:5 n3 and C22:6 n3. Of those screened, only ten fungi were capable of VLCPUFA production, with Mortierella the predominant VLCPUF A producing species. Four Oomycete species were also capable of VLCPUF A production. It is thought that only basal fungal lineages, such as species from the Chytridiomycota and Zygomycota, are capable of VLCPUF A production. It was also found that VLCPUF As are not essential for growth at low temperatures, as Penicillium rugulosum, capable of producing 'fatty acids no longer or more unsaturated than C18:3 n3, demonstrated over 2 g of biomass per 100 ml of broth when grown at 5°C. This indicates that trienoic fatty acids are sufficient for maintaining membrane fluidity, although other factors may play a role in P. rugulosum's low temperature growth. Comparatively, VLCPUFA producing Mortierella species produced 200-250 mg of biomass, whereas the majority of non- VLCPUF A producing isolates produced 106-115 mg of biomass per 100 ml of broth. The total lipid unsaturation indices of nine isolates grown under three temperature regimes showed that the lowest growth temperature, 5°C, produced the highest un saturation index value III SIX of the organisms. 15°C produced the highest unsaturation index value in two of the isolates. This suggests that temperature has an effect on fungal lipid composition, and that lower temperatures may increase lipid unsaturation levels. It was also found that the ~6 elongase, initially identified from Mortierella alpina, is indicative for VLCPUF A producing fungi. The genomic conserved sequence found within ~6 elongases was used to develop primer sets that could be used with a PCR based methodology to screen fungal isolates for VLCPUF A production. The method successfully identified VLCPUF A producing Mortierella and Allomyces species, and was not found to amplify non-Ad elongases. Finally, recombinantPhaffia rhodozyma strains were developed using the ~5 desaturase and ~6 elongase from Mortierella alpina. The fatty acid profiles of the recombinant strains displayed novel fatty acids such as C20:2 n6 and C20:3 n3, and putatively, C18:2 ~5, 9 and C18:3 ~5, 9, 12 which correlated with the inserted genes.

L’adaptation à la vie extra-utérine requiert la résorption rapide du liquide pulmonaire, la création de la capacité fonctionnelle résiduelle (FRC), ainsi que l’augmentation du débit pulmonaire. Bien que ces mécanismes aient été largement étudiés, leurs cinétiques restent mal connues faute de méthode d’étude non invasive in vivo. L’échec de l’adaptation cardiorespiratoire à la naissance est la principale cause de mortalité et de morbidité de la période néonatale, malgré l’utilisation de stratégie préventive comme la corticothérapie anténatale, et l’amélioration des techniques de réanimation néonatale. Les acides gras polyinsaturés oméga 3 (AGPI n3) que l’on retrouve dans les huiles de poisson ont des effets cardiovasculaires et respiratoires qui pourraient améliorer la transition périnatale. Par ailleurs, leurs propriétés anti-inflammatoires peuvent réduire les conséquences des effets iatrogènes des techniques de réanimation dont la mise en oeuvre est nécessaire en cas de maladaptation cardiorespiratoire à la naissance. Les objectifs de ce travail ont été de : Mettre au point et valider une méthode d’exploration de la cinétique de réabsorption du liquide pulmonaire à la naissance par résonnance magnétique nucléaire Etudier les effets des AGPI n3 sur : l’adaptation ventilatoire à la naissance la circulation pulmonaire périnatale la prévention des lésions pulmonaires induites par une hyperoxie prolongée Les études ont été réalisées sur les modèles expérimentaux de foetus de brebis et de ratons nouveau-nés exposés à une hyperoxie prolongée. Nous montrons que la technique par IRM permet d’évaluer la cinétique de clairance du liquide pulmonaire in vivo en situations physiologique et pathologique. Alors que les AGPI n3 améliorent la circulation pulmonaire, ils n’ont pas d’effet sur la clairance du liquide pulmonaire. Par contre, ils réduisent les lésions pulmonaires induites par l’oxygène. Ces résultats expérimentaux suggèrent que les AGPI n3 pourraient prévenir les échecs d’adaptation cardiorespiratoire à la naissance et ses conséquences à long terme. / Perinatal transition requires clearance of pulmonary fluid, creation of functional respiratory capacity (FRC), and the multiplication of pulmonary blood flow by ten, to ensure gas exchange and blood oxygenation. Physiology of perinatal transition has been broadly studied but the kinetics of its evolution is less known. The aim of our study was to assess the kinetics of the respiratory transition. Besides, we supposed that polyunsaturated fatty-acids omega 3 (n3 PUFA) would improve this transition. To study the kinetic of respiratory transition, we used the MRI in 3 groups of lambs: premature group (Preterm) with a term of 122-124 days, two groups of late preterm lambs (134-136 days), including one group who received antenatal steroids (Late preterm + steroids), and a group without steroids (Late preterm). Surprisingly, we observed a rapid increase of lung fluid in preterm lambs reaching 30% at the end of the experimentation. Creation of FRC was also impaired in this group. In the Late preterm group, clearance of pulmonary fluid was slowed down with a moderate fall of 10%, whereas, in the Late preterm + steroids group, lung fluid dropped by 50% (p<0.05). Similarly, FRC in the Late preterm + steroids group was higher than the other 2 groups (p <0.05). Respiratory functions were closely related to lung fluid content and to FRC. We hypothesized that n3 PUFA would improve perinatal transition using two models: - An acute model: in a chronically instrumented model, catheters were introduced in the jugular and the carotid vessels of lamb fetuses at 122 days of gestation. Lamb fetuses received either Omegaven (n3 PUFA), or Intralipide (n6 PUFA), or isotonique physiological serum (SSI) for 4 days. Respiratory transition was explored by MRI. There was no significant difference between these three groups. Total lung fluid increased by about 20% in Omegaven group and 18% in SSI group (p> 0.05). - A model of Bronchopulmonary dysplasia: pups were exposed to hyperoxia for 10 days, and feeding rats received supplementation by either n3 PUFA (Omacor), n6 PUFA (sunflower oil), or SSI, with control groups that received the same feeding supply but which were exposed to air. Histological studies showed bronchopulmonary dysplasia lesions in the Hyperoxia groups, characterized by decrease of alveolar number, decrease in the number of secondary alveolar septa, and widening of the interstitial space. These lesions were similar in SSI and n6 PUFA groups, whereas n3 PUFA improved these lesions (p <0.05). To assess the effects of n3 PUFA in pulmonary vascular resistances (PVR), we studied their effects on fetal circulation characterized by elevated RVP using a chronically instrumented experimental model of lamb fetus. Lamb fetuses received either n3 PUFA (Omegaven), or n6 PUFA (Intralipid). We then explored the action mechanisms of n3 PUFA: 1) NO pathway using L-Nitro Argenine (LNA), 2) potassium channels pathway using Tetraethylamonium (TEA) and 3) epoxides produced by cytochrome P450 using MS-PPOH. Finally, we investigated the effect of Eicosapentaenoic Acid (EPA) perfusion, or Docosahexaenoic Acid (DHA) perfusion in pulmonary circulation. We showed that, unlike Intralipid, Omegaven induced pulmonary vasodilation, and an increase in pulmonary flow. An identical effect was reproduced by EPA infusion, while DHA did not modify PVR. This response was not modified by LNA. But it was reduced by MS-PPOH, and abolished by TEA. These results indicate that n3 PUFA induced pulmonary vasodilation, mediated by epoxides which act on potassium channels. To conclude, we demonstrated that respiratory transition in preterm lambs even in late preterm ones is altered with the presence of pulmonary edema. n3 PUFA did not improve lung fluid clearance but should be considered in the tretment of pulmonary hypertension, and in the prevention of brochopulmonary dysplasia of the newborns.

Background Iron deficiency (ID) is the world‟s most prevalent micronutrient deficiency and predominantly affects developing countries, also South Africa. In areas with low fish consumption and high n-6 PUFA vegetable oil intake, there is a risk for having inadequate n-3 PUFA status. Both iron and n-3 PUFA play important roles in the immune response, and supplementation is a strategy to alleviate deficiencies. However, little is known about potential interactive effects between concurrent iron and n-3 PUFA supplementation on the immune system. This is also important in the context that iron supplementation may be unsafe and may increase morbidity and mortality. Aim The overall aim of this thesis was to assess the effects of iron and docosahexaenoic (DHA)/eicosapentaenoic acid (EPA) supplementation, alone and in combination, on the immune system of ID children. More specifically, these effects were investigated on the occurrence and duration of illness and school-absenteeism due to illness, peripheral blood mononuclear cell (PBMC), red blood cell (RBC) and plasma total phospholipid fatty acid composition, iron status, fatty acid-derived immune modulators and targeted PBMC gene expression. Furthermore, association of PBMC, RBC and plasma total phospholipid fatty acid composition with allergic disease, were also examined. Design In a 2-by-2 factorial, randomised, double-blind, placebo-controlled trial, South African children (n = 321, aged 6–11 y) were randomly assigned to receive oral supplements of either 1) iron (50 mg as ferrous sulphate) plus placebo; 2) DHA/EPA (420/80 mg) plus placebo; 3) iron plus DHA/EPA (420/80 mg); or 4) placebo plus placebo for 8.5 mo, four times per week. Absenteeism and illness symptoms were recorded and biochemical parameters for compliance as well as parameters fundamental to immune function were assessed at baseline and endpoint. Furthermore, in a cross-sectional design, associations of allergic disease with baseline fatty acid composition of PBMC, RBC and plasma were examined. Results The combination of iron and DHA/EPA significantly attenuated respiratory illness caused by iron supplementation. DHA/EPA supplementation alone improved respiratory symptoms at school, but increased headache-related absenteeism. DHA/EPA and iron supplementation individually tended to increase and decrease anti-inflammatory DHA and EPA-derived mediators, respectively. Furthermore the anti-inflammatory DHA-derived immune mediator, 17HDHA was higher in the DHA/EPA plus placebo and iron plus DHA/EPA groups than in the iron plus placebo group. Also, the pro-inflammatory arachidonic acid (AA)-derived modulators (5- and 15-hydroxyeicosapentaenoic acid) were significantly lower in the iron plus DHA/EPA group compared to the placebo plus placebo groups. In the study population, 27.2% of the children had allergic disease and AA in PBMC phospholipids was significantly lower in the allergic children than in the non-allergic children. In RBC phospholipids dihomo-gamma-linolenic acid (DGLA) and the ratio of DGLA: linoleic acid (LA) correlated negatively and the n-6:n-3 PUFA ratio positively with total immunoglobulin E (tIgE). Furthermore, trans-C18:1n-9, tended to be higher in the allergic group. Conclusion DHA/EPA prevented respiratory illness caused by iron supplementation and although DHA/EPA on its own reduced respiratory morbidity when the children were present at school, surprisingly it increased the likelihood of being absent with headache and fever. The biochemical findings compliment the clinical results and support previous observations about DHA/EPA supplementation to reduce inflammation, but add to the current knowledge base that a relatively high oral dose of non-haem iron modulates circulating lipid-derived immune modulators and related gene expression. Furthermore, when supplementing with iron and DHA/EPA combined, in this ID population with low fish intake, the anti-inflammatory effect of DHA/EPA is maintained concurrently with attenuation of respiratory morbidity. This finding support the notion that excess iron (probably as non-transferrin bound iron) becomes available for pathogens and is probably why we found that iron increased respiratory infectious morbidity. The improved clinical outcome with combined supplementation seems to be related to increased lipid-mediator synthesis gene expression and the availability of DHA/EPA, leading to a more pro-resolving profile and enhanced immune competence. Overall these results give better insight into immune function and infectious morbidity in relation to n-3 PUFA and iron status and treatment, as well as the possible association of fatty acid status with allergic disease in young South-African school children. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

Background Iron deficiency (ID) is the world‟s most prevalent micronutrient deficiency and predominantly affects developing countries, also South Africa. In areas with low fish consumption and high n-6 PUFA vegetable oil intake, there is a risk for having inadequate n-3 PUFA status. Both iron and n-3 PUFA play important roles in the immune response, and supplementation is a strategy to alleviate deficiencies. However, little is known about potential interactive effects between concurrent iron and n-3 PUFA supplementation on the immune system. This is also important in the context that iron supplementation may be unsafe and may increase morbidity and mortality. Aim The overall aim of this thesis was to assess the effects of iron and docosahexaenoic (DHA)/eicosapentaenoic acid (EPA) supplementation, alone and in combination, on the immune system of ID children. More specifically, these effects were investigated on the occurrence and duration of illness and school-absenteeism due to illness, peripheral blood mononuclear cell (PBMC), red blood cell (RBC) and plasma total phospholipid fatty acid composition, iron status, fatty acid-derived immune modulators and targeted PBMC gene expression. Furthermore, association of PBMC, RBC and plasma total phospholipid fatty acid composition with allergic disease, were also examined. Design In a 2-by-2 factorial, randomised, double-blind, placebo-controlled trial, South African children (n = 321, aged 6–11 y) were randomly assigned to receive oral supplements of either 1) iron (50 mg as ferrous sulphate) plus placebo; 2) DHA/EPA (420/80 mg) plus placebo; 3) iron plus DHA/EPA (420/80 mg); or 4) placebo plus placebo for 8.5 mo, four times per week. Absenteeism and illness symptoms were recorded and biochemical parameters for compliance as well as parameters fundamental to immune function were assessed at baseline and endpoint. Furthermore, in a cross-sectional design, associations of allergic disease with baseline fatty acid composition of PBMC, RBC and plasma were examined. Results The combination of iron and DHA/EPA significantly attenuated respiratory illness caused by iron supplementation. DHA/EPA supplementation alone improved respiratory symptoms at school, but increased headache-related absenteeism. DHA/EPA and iron supplementation individually tended to increase and decrease anti-inflammatory DHA and EPA-derived mediators, respectively. Furthermore the anti-inflammatory DHA-derived immune mediator, 17HDHA was higher in the DHA/EPA plus placebo and iron plus DHA/EPA groups than in the iron plus placebo group. Also, the pro-inflammatory arachidonic acid (AA)-derived modulators (5- and 15-hydroxyeicosapentaenoic acid) were significantly lower in the iron plus DHA/EPA group compared to the placebo plus placebo groups. In the study population, 27.2% of the children had allergic disease and AA in PBMC phospholipids was significantly lower in the allergic children than in the non-allergic children. In RBC phospholipids dihomo-gamma-linolenic acid (DGLA) and the ratio of DGLA: linoleic acid (LA) correlated negatively and the n-6:n-3 PUFA ratio positively with total immunoglobulin E (tIgE). Furthermore, trans-C18:1n-9, tended to be higher in the allergic group. Conclusion DHA/EPA prevented respiratory illness caused by iron supplementation and although DHA/EPA on its own reduced respiratory morbidity when the children were present at school, surprisingly it increased the likelihood of being absent with headache and fever. The biochemical findings compliment the clinical results and support previous observations about DHA/EPA supplementation to reduce inflammation, but add to the current knowledge base that a relatively high oral dose of non-haem iron modulates circulating lipid-derived immune modulators and related gene expression. Furthermore, when supplementing with iron and DHA/EPA combined, in this ID population with low fish intake, the anti-inflammatory effect of DHA/EPA is maintained concurrently with attenuation of respiratory morbidity. This finding support the notion that excess iron (probably as non-transferrin bound iron) becomes available for pathogens and is probably why we found that iron increased respiratory infectious morbidity. The improved clinical outcome with combined supplementation seems to be related to increased lipid-mediator synthesis gene expression and the availability of DHA/EPA, leading to a more pro-resolving profile and enhanced immune competence. Overall these results give better insight into immune function and infectious morbidity in relation to n-3 PUFA and iron status and treatment, as well as the possible association of fatty acid status with allergic disease in young South-African school children. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015