Indiana University-Purdue University Indianapolis (IUPUI) / Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distr ibuted to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. T his research employs model membranes of well - defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega - 3 (n - 3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect they have on molecular organization may be, in part, responsible. Chol is a major constituent in the plasma membrane of mammals. It determines the arrangement and collective properties of neighboring lipids, driving the formation of domains via differential affinity for different lipids . T he m olecular organization of 1 -[ 2 H 31 ]palmitoyl -2- eicosapentaenoylphosphatidylcholine (PEPC - d 31 ) and 1 -[ 2 H 31 ]palmitoyl -2- docosahexaenoylphosphatidylcholine (PDPC -d 31 ) in membran es with sphingomyelin (SM) and chol (1:1:1 mol) was compared by solid - state 2 H NMR spectroscopy. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n - 3 PUFAs found in fish oil, while PEPC -d 31 and PDPC -d 31 are phospholipids containing the respective PUFAs at the sn - 2 position and a perdeuterated palmitic acid a t the sn - 1 position . Analysis of s pectra recorded as a function of temperature indicate s that in both cases, formation of PUFA - rich (less ordered) and SM - rich (more ordered) domains occurred. A surprisingly substantial proportion of PUFA was found to infil trate the more ordered domain. There was almost twice as much DHA (65%) as EPA (30%) . The implication is that n - 3 PUFA s can incorporate into lipid rafts, which are domains enriched in SM and chol in the plasma membrane, and potentially disrupt the activity of signaling proteins that reside therein. DHA, furthermore, may be the more potent component of fish oil. PUFA - chol interactions were also examined through affinity measurements. A novel method utilizing electron paramagnetic resonance (EPR) was develope d, to monitor the partitioning of a spin - labeled analog of chol , 3β - doxyl - 5α - cholestane (chlstn), between large unilamellar vesicles (LUVs) and met hyl - β - cyclodextrin (mβCD). The EPR spectra for chlstn in the two environments are distinguishable due to the substantial differences in tumbling rates , allowing the population distribution ratio to be determined by spectral simulation. Advantages of this approach include speed of implementation and a vo idance of potential artifact s associated with physical separation of LUV and mβCD . Additionally, in a check of the method, t he relative partition coefficients between lipids measured for the spin label analog agree with values obtained for chol by isothermal titration calorimetry (ITC). Results from LUV with different composition confirmed a hierarchy of decreased sterol affinity for phospholipids with increasing acyl chain unsaturation , PDPC possessing half the affinity of the corresponding monounsaturated phospholipid. Taken together, the results of these studies on model membranes demonstrate the potential for PUFA - driven alteration of the architecture of biomembranes, a mechanism through which human health may be impacted.

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

Contexte : La dysfonction du tissu adipeux blanc (TAB) favorise les facteurs de risque de diabète de type 2 (DbT2), c’est-à-dire la résistance à l’insuline (RI), l’hyper sécrétion d’insuline glucostimulée (SIGS), le délai de clairance des gras et les concentrations élevées d’apoBlipoprotéines (apoB plasmatique) incluant les lipoprotéines de faible densité (LDL). De récentes études de notre laboratoire et d’autres suggèrent que le niveau élevé d’apoB plasmatique (hyperapoB) est une cause et non seulement une conséquence de la dysfonction du TAB. De plus, une internalisation augmentée d’apoB-lipoprotéines via les récepteurs tels que le récepteur aux LDLs (LDLR) et le cluster de différenciation 36 (CD36), favorise le risque de DbT2. Cependant, les mécanismes sous-jacents de même que les interventions nutritionnelles pour les cibler demeurent incertains. L'activation de la voie de l’inflammasome NLRP3/ interleukine (IL) -1β favorise la dysfonction du TAB et les facteurs de risque de DbT2 et est activée par les LDLs oxydées dans les cellules immunitaires. L'acide eicosapentaénoïque (AEP) et l'acide docosahexaénoïque (ADH) réduisent l'hyperapoB, l'activité de l’inflammasome NLRP3 dans les cellules immunitaires et les facteurs de risque de DbT2 chez l’humain. Ils sont synthétisés de façon endogène par l’entremise des désaturases d’acides gras δ-5 (D5D) et δ-6 (D6D). Chez l’humain, de faibles niveaux d’AEP et d’ADH circulants et d’activité de la D5D et une activité élevée de la D6D prédisent l'incidence de DbT2 et la RI par des mécanismes inconnus. Objectifs : L'hypothèse de ma thèse est que l'AEP et l’ADH améliorent les facteurs de risque de DbT2, soit la dysfonction du TAB, le délai de clairance des gras, la RI et l’hyper SIGS, ceci via une baisse de l'apoB plasmatique et de l’activité de l’inflammasome NLRP3 dans le TAB. Les objectifs sont d'examiner si: 1) les associations entre les activités de la D5D et de la D6D et les facteurs de risque de DbT2 dépendent de l'apoB plasmatique; 2) la supplémentation en AEP+ADH réduit l'apoB plasmatique, l'expression du LDLR et du CD36 dans le TAB, l'activité de l’inflammasome NLRP3 dans le TAB et les facteurs de risque de DbT2; 3) l’AEP+ADH inhibe la sécrétion d'IL-1β par le TAB humain stimulée par des signaux canoniques ou les LDLs natives. Méthodes: Des hommes et des femmes postménopausées normoglycémiques ont été testés à l’état basal et après une supplémentation en AEP (1,8 g/jour) et ADH (0,9 g/jour) de 12 semaines. Les activités de la D5D et de la D6D ont été estimées à partir des acides gras produits/précurseurs dans les phospholipides plasmatiques. Nous avons mesuré la SIGS, la RI et le disposition index lors d’un clamp Botnia. Après un repas à 66% de gras, le délai de clairance des gras a été mesuré par l’aire sous la courbe (sur 6 h) des triglycérides (TG) ou de l’apoB48 (chylomicrons) plasmatiques. Ex vivo dans une biopsie de TAB, nous avons mesuré l'expression de surface du LDLR et du CD36 par immunohistochimie, l'ARNm de NLRP3 et IL1B par RT-qPCR et la sécrétion d'IL-1β par alpha-LISA en l’absence ou en présence d’une stimulation par le lipopolysaccharide (LPS), l'adénosine triphosphate (ATP) et/ou les LDLs humaines natives et lors d’une co-incubation avec l’AEP+ADH. Résultats: À l’état basal (N=98), l'activité de la D5D corrélait négativement avec l'apoB plasmatique, la 2e phase de SIGS, la RI et le délai de clairance des chylomicrons et ces associations étaient dépendantes de l'apoB plasmatique. Inversement, l'activité de la D6D corrélait positivement avec la SIGS, la RI et le délai de clairance des chylomicrons indépendamment de l'apoB plasmatique. Chez les sujets ayant complété la supplémentation en AEP+ADH (N=30), on notait une amélioration de la 1e phase de SIGS, du disposition index et de la clairance des TGs. Des niveaux initiaux plus élevés d'apoB plasmatique, de TGs postprandiaux plasmatiques et de RI, et dans le TAB d'expression du LDLR et du CD36, de sécrétion d’IL-1β et d'ARNm de NLRP3 prédisaient une plus grande réduction de ces paramètres. En comparaison à l'acide palmitique, l’AEP+ADH inhibait la sécrétion d'IL-1β par le TAB, en l’abscence ou en présence d’une stimulation par le LPS, l'ATP et/ou les LDLs natives de ces sujets. Conclusion: Les associations inverses entre l'activité de la D5D avec les facteurs de risque de DbT2 sont dépendantes de l'apoB plasmatique. Les meilleurs répondants à la supplémentation en AEP et ADH, en termes de réduction d'apoB plasmatique, d’expression du LDLR et du CD36 dans le TAB, d'activité de l’inflammasome NLRP3 dans le TAB, de TGs postprandiaux et de RI, sont les sujets avec des niveaux initiaux élevés de ces paramètres. L’AEP et l’ADH inhibent directement la sécrétion d'IL-1β par le TAB humain induite par les LDLs natives ou d'autres signaux. Nous proposons que la supplémentation en AEP et ADH puisse cibler l'activité de l’inflammasome NLRP3 dans le TAB, induite par un niveau élevé d’apoB-lipoprotéines plasmatiques ou internalisées par les récepteurs, et ainsi aider à prévenir le DbT2. / Background: White adipose tissue (WAT) dysfunction promotes risk factors for type 2 diabetes (T2D), namely insulin resistance (IR), high glucose-stimulated insulin secretion (GIIS), delayed fat clearance and high concentrations of apoB-lipoproteins (measured as plasma apoB) including low density lipoproteins (LDL). Recent studies from our lab and others suggest that high plasma apoB (hyperapoB) is a cause and not only a consequence of WAT dysfunction. Moreover, upregulated receptor-mediated uptake of apoB-lipoproteins via LDL receptor (LDLR) and cluster of differentiation 36 (CD36), promotes the risk for T2D. However, underlying mechanisms as well as nutritional interventions to target them remain unclear. Activation of the NLRP3 inflammasome/interleukin (IL)-1β pathway promotes WAT dysfunction and risk factors for T2D and is activated by oxidized LDLs in immune cells. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) reduce hyperapoB, NLRP3 inflammasome activity in immune cells and risk factors for T2D in humans. They are synthesized endogenously through δ-5 (D5D) and δ-6 (D6D) fatty desaturases. In humans, low levels of circulating EPA and DHA and D5D activity and high D6D activity predict the incidence of T2D and IR by unknown mechanisms. Objectives: The hypothesis of my thesis is that EPA and DHA improve T2D risk factors, namely WAT dysfunction, delayed fat clearance, IR and high GIIS, this via a reduction of plasma apoB and WAT NLRP3 inflammasome activity. The objectives are to examine whether: 1) the associations between the levels of D5D and D6D activities and the risk factors for T2D are dependent on plasma apoB; 2) supplementation with EPA+DHA reduces plasma apoB, WAT LDLR and CD36 expression, WAT NLRP3 inflammasome activity and T2D risk factors; 3) EPA+DHA directly inhibits IL-1β secretion from human WAT stimulated by canonical signals or native LDLs. Methods: Normoglycemic men and postmenopausal women were tested at baseline and after supplementation with EPA (1.8 g/day) and DHA (0.9 g/day) for 12 weeks. The activities of D5D and D6D were estimated from the product/precursor fatty acids in plasma phospholipids. We measured GIIS, IR and disposition index by a Botnia clamp. Following a 66% fat meal, delayed fat clearance was measured as the area under the curve (over 6 h) of plasma triglycerides (TG) or apoB48 (chylomicrons). Ex vivo in a WAT biopsy, we measured LDLR and CD36 surface expression by immunohistochemistry, NLRP3 and IL1B mRNA by RT-qPCR, and IL-1β secretion by alpha-LISA either unstimulated or stimulated by lipopolysaccharide (LPS), adenosine triphosphate (ATP), and/or native human LDLs, and during co-incubation with EPA+DHA. Results: At baseline (N=98), D5D activity correlated negatively with plasma apoB, 2nd phase GIIS, IR and delayed chylomicron clearance and these associations were dependent on plasma apoB. Conversely, D6D activity correlated positively with GIIS, IR, and delayed chylomicron clearance independently of plasma apoB. In subjects who completed the EPA+DHA supplementation (N=30), there was an amelioration in 1st phase GIIS, disposition index and TG clearance. Higher baseline levels of plasma apoB, plasma postprandial TGs, IR, WAT LDLR and CD36 surface expression, WAT IL-1β secretion and WAT NLRP3 mRNA predicted a greater reduction of these parameters. In comparison with palmitic acid, EPA+DHA inhibited IL-1β secretion from WAT, either unstimulated or stimulated by LPS, ATP and/or subjects’ native LDLs. Conclusion: The negative associations of D5D activity with risk factors for T2D are dependent on plasma apoB. Best responders to EPA and DHA supplementation to reduce plasma apoB, WAT LDLR and CD36 expression, WAT NLRP3 inflammasome activity, delayed TG clearance, and IR are subjects with elevated baseline levels of these parameters. EPA and DHA directly inhibit IL-1β secretion from human WAT induced by native LDLs or other signals. We propose that EPA and DHA supplementation may target upregulated WAT NLRP3 inflammasome activity induced by high plasma concentrations, or receptor-mediated uptake, of apoB-lipoproteins, and thus help prevent T2D.

Tesis por compendio / [ES] El rápido crecimiento de la acuicultura ha generado una serie de problemas relacionados con la disponibilidad de ingredientes marinos esenciales en la alimentación de peces, debido a su alto contenido en ácidos grasos poliinsaturados de cadena larga (LC-PUFAs) omega-3. Una estrategia altamente innovadora para la obtención de nuevos ingredientes ricos en LC-PUFAs, consiste en el cultivo intensivo de organismos que sean capaces de producirlos a partir de fuentes que carecen de éstos, como son los subproductos de industrias agroalimentarias. Este enfoque requiere entender la capacidad biosintética del organismo en cuestión, y cómo ésta se puede modular para aumentar la acumulación de estos compuestos. Esta Tesis Doctoral plantea una serie de estudios dirigidos a esclarecer el potencial de los gamáridos para aplicar esta estrategia. La biosíntesis de LC-PUFAs en animales requiere de la acción coordinada de enzimas elongasas y desaturasas, implicadas en la conversión de ácidos grasos relativamente cortos y poco insaturados, en LC-PUFAs de alto valor nutricional. Así pues, esta Tesis Doctoral ha investigado, por un lado, la presencia y la actividad de genes involucrados en la biosíntesis de LC-PUFAs en gamáridos tanto marinos como dulceacuícolas. Por otro lado, ha estudiado cómo diferentes dietas y temperaturas afectan al metabolismo de ácidos grasos, crecimiento y supervivencia, en el gamárido marino Gammarus locusta, tanto a nivel fisiológico y composicional, como a nivel molecular. Los resultados de la primera parte demuestran que los gamáridos tienen varias elongasas, pero carecen de desaturasas. Se identificaron tres tipos de elongasas (elovl4, elovl6, y elovl1/7-like) en el gamárido marino Echinogammarus marinus, siendo elovl4 y elovl1/7-like clave para la elongación de LC-PUFAs. Este mismo patrón de elongasas se repite también para los gamáridos dulceacuícolas. Sorprendentemente, se han encontrado desaturasas front-end (fed), methyl-end (wx-des), y una elongasa del tipo Elovl2/5 únicamente en transcriptomas de gamáridos dulceacuícolas. Posteriores análisis moleculares y filogenéticos han podido determinar que estas secuencias fed, wx-des, y elovl2/5 son propias de rotíferos bdeloideos, epibiontes de gamáridos dulceacuícolas. Estos hallazgos, más allá de apuntar el potencial biosintético de los rotíferos bdeloideos, revela una relación constante entre éstos y los gamáridos en ecosistemas dulceacuícolas. La segunda parte de la Tesis analiza cómo dieta y temperatura modulan el metabolismo de ácidos grasos, crecimiento y supervivencia en Gammarus locusta. Los resultados han mostrado que G. locusta experimenta un mayor crecimiento, aunque también mayores tasas de mortalidad, cuando se cultiva a temperaturas elevadas. La mortalidad en G. locusta también aumenta cuando este se alimenta con una dieta rica en ácidos grasos saturados (SFAs) y carente de LC-PUFAs. Los perfiles de ácidos grasos en gamáridos reflejan las dietas consumidas, independientemente de la temperatura; aunque cabe destacar que los gamáridos alimentados con dietas carentes de LC-PUFAs conservan niveles apreciables de estos compuestos en sus lípidos corporales. A nivel molecular, una dieta sin LC-PUFAs y rica en SFAs disminuye el catabolismo de ácidos grasos y favorece su acumulación modulando la expresión de genes involucrados en estos procesos. Además, estas mismas condiciones afectan negativamente el desarrollo y supervivencia al influir en genes relacionados con el ciclo de muda. Además, se ha observado que temperaturas altas aceleran el desarrollo e incrementan la mortalidad. Según estos resultados, puede concluirse que las temperaturas elevadas y dietas carentes de LC-PUFAs no son adecuadas para el cultivo de G. locusta cuando el objetivo final es la obtención de biomasa rica en estos compuestos, ya que dichas condiciones afectan negativamente al perfil nutricional del animal y a su supervivencia. / [CA] El ràpid creixement de l'aqüicultura ha generat una sèrie de problemes relacionats amb la disponibilitat d'ingredients marins essencials en l'alimentació de peixos, a causa del seu alt contingut en àcids grassos poliinsaturats de cadena llarga (LC-PUFAs) omega-3. Una estratègia altament innovadora per a l'obtenció de nous ingredients rics en LC-PUFAs, consistix en el cultiu intensiu d'organismes que siguen capaços de produir-los a partir de fonts que no els ténen, com són els subproductes d'indústries agroalimentàries. Aquesta estratègia requerix entendre la capacitat biosintètica de l'organisme en qüestió, i com aquesta es pot modular per a augmentar l'acumulació d'estos compostos. Esta Tesi Doctoral planteja una sèrie d'estudis dirigits a esclarir el potencial dels gammàrids per a aplicar esta estratègia. La biosíntesi de LC-PUFAs en animals requerix de l'acció coordinada d'enzims elongases i desaturases, implicats en la conversió d'àcids grassos relativament curts i poc insaturats, en LC-PUFAs d'alt valor nutricional. Així doncs, esta Tesi Doctoral ha investigat, d'una banda, la presència i l'activitat de gens involucrats en la biosíntesi de LC-PUFAs en gammàrids tant marins com dulciaqüícoles. D'altra banda, ha estudiat com diferents dietes i temperatures afecten el metabolisme d'àcids grassos, creixement i supervivència, en el gammàrid marí Gammarus locusta, tant a nivell fisiològic i composicional, com a nivell molecular. Els resultats de la primera part demostren que els gammàrids ténen diverses elongases, però manquen de desaturases. Es van identificar tres tipus d'elongases (elovl4, elovl6, i elovl1/7-like) en el gammàrid marí Echinogammarus marinus, sent elovl4 i elovl1/7-like clau per a l'elongació de LC-PUFAs. Este mateix patró d'elongases es repetix també per als gammàrids dulciaqüícoles. Sorprenentment, s'han trobat desaturases front-end (fed), methyl-end (wx-des), i una elongasa del tipus Elovl2/5 únicament en transcriptomes de gammàrids dulciaqüícoles. Posteriors anàlisis moleculars i filogenètics han pogut determinar que estes seqüències fed, wx-des, i elovl2/5 són pròpies de rotífers bdeloides, epibionts de gammàrids dulciaqüícoles. Estes troballes, més enllà d'apuntar el potencial biosintètic dels rotífers bdeloides, revela una relació constant entre estos i els gammàrids en ecosistemes dulciaqüícoles. La segona part de la Tesi analitza com dieta i temperatura modulen el metabolisme d'àcids grassos, creixement i supervivència en Gammarus locusta. Els resultats han mostrat que G. locusta experimenta un major creixement, encara que també majors taxes de mortalitat, quan es cultiva a temperatures elevades. La mortalitat en G. locusta també augmenta quan este s'alimenta amb una dieta rica en àcids grassos saturats (SFAs) i sense LC-PUFAs. Els perfils d'àcids grassos en gammàrids reflectixen les dietes consumides, independentment de la temperatura; encara que cal destacar que els gamàrids alimentats amb dietes mancants de LC-PUFAs conserven nivells apreciables d'estos compostos en els seus lípids corporals. A nivell molecular, una dieta sense LC-PUFAs i rica en SFAs disminuïx el catabolisme d'àcids grassos i afavorix la seua acumulació modulant l'expressió de gens involucrats en estos processos. A més, estes mateixes condicions afecten negativament el desenvolupament i supervivència al influir en gens relacionats amb el cicle de muda. A més, s'ha observat que temperatures altes acceleren el desenvolupament i incrementen la mortalitat. Segons estos resultats, pot concloure's que les temperatures elevades i dietes mancants de LC-PUFAs no són adequades per al cultiu de G. locusta quan l'objectiu final és l'obtenció de biomassa rica en estos compostos, ja que estes condicions afecten negativament el perfil nutricional de l'animal i a la seua supervivència. / [EN] The rapid growth of aquaculture has generated a series of problems related to the availability of essential marine ingredients in fish feed, due to their high content of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs). A highly innovative strategy for obtaining new ingredients rich in LC-PUFAs relies on the intensive culture of organisms that are capable of producing LC-PUFAs from sources that lack them, such as the by-products of agri-food industries. This approach requires understanding the biosynthetic capacity of the organism, and how this can be modulated to increase the accumulation of these compounds. This Doctoral Thesis proposes a series of studies aimed at clarifying the potential of gammarids to apply this strategy. LC-PUFA biosynthesis requires the coordinated action of elongase and desaturase enzymes, involved in the conversion of relatively short and poorly unsaturated fatty acids into LC-PUFAs of high nutritional value. Thus, this Doctoral Thesis has investigated, on one hand, the presence and activity of genes involved in the LC-PUFAs biosynthesis in both marine and freshwater gammarids. On the other hand, we have studied how different diets and temperatures affect fatty acid metabolism, growth and survival, in the marine gammarid Gammarus locusta, both at a physiological and compositional level, and at a molecular level. The results of the first part demonstrate that gammarids have several elongases, but lack desaturases. Three types of elongases (elovl4, elovl6, and elovl1/7-like) were identified in the marine gammarid Echinogammarus marinus, being elovl4 and elovl1/7-like key for LC-PUFA elongation. This same pattern of elongases is also present in their freshwater counterparts. Surprisingly, front-end desaturases (fed), methyl-end (wx-des), and an Elovl2/5-type elongase have been found only in transcriptomes built from freshwater gammarids. Subsequent molecular and phylogenetic analyses have been able to determine that these fed, wx-des, and elovl2/5 sequences are typical of bdelloid rotifers, which are epibionts of freshwater gammarids. These findings, beyond pointing out the biosynthetic potential of bdelloid rotifers, reveal a constant relationship between them and gammarids in freshwater ecosystems. The second part of the Thesis focuses on how diet and temperature modulate fatty acid metabolism, growth and survival in Gammarus locusta. The results have shown that G. locusta experiences greater growth, but also higher mortality rates, when grown at higher temperatures. Mortality in G. locusta also increases when it is fed with a diet rich in saturated fatty acids (SFAs) and lacking LC-PUFAs. Fatty acid profiles in gammarids reflect the diets consumed, regardless of temperature; although it is worth noting that gammarids fed with diets lacking LC-PUFAs still show levels of these compounds in their body lipids. At a molecular level, a diet lacking LC-PUFAs and rich in SFAs decreases the catabolism of fatty acids and enhances their accumulation by modulating the expression of genes involved in these processes. Furthermore, these same conditions negatively affect development and survival by influencing genes related to the molting cycle. In addition, it has been observed that higher temperatures accelerate development and increase mortality. According to these results, it can be concluded that higher temperatures and diets lacking LC-PUFAs are not suitable for the culture of G. locusta when the final outcome is to obtain a biomass rich in these compounds, since these conditions negatively affect the nutritional profile of the animal and its survival. / This research was supported by the ERA-NET BlueBio COFUND Project SIDESTREAM (Grant ID 68), co-funded through national funds provided by the Agencia Estatal de Investigación, Spain, grant no. PCI2020-111960/MCIN/AEI/10.13039/501100011033, the German Federal Ministry of Education and Research (BMBF), FKZ161B0950B, and the Fundação para a Ciência e a Tecnologia, Por- tugal (BLUEBIO/0005/2019). Additional funding was received through the project IMPROMEGA Agencia Española de Investigación, Spain, grant no. RTI2018-095119-B-100, MCIU/AEI/FEDER/UE/ MCIN/AEI/10.13039/501100011033/ and FEDER ‘A way to make Europe / Ribes Navarro, A. (2024). Biosíntesis de ácidos grasos poliinsaturados de cadena larga en gamáridos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/207006 / Compendio