Hyperglycemia in critically ill patients was considered for many years an adaptive response to stress conditions being present in both patients with and without previous history of diabetes. Hyperglycemia is caused mainly by peripheral insulin resistance induced by the factors acting counteracting insulin signalling at the postreceptor level. Furthermore, hyperglycemia itself can then increase serum levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (Il-6) and interleukin-8 (Il- 8) and others. On the contrary, peripheral insulin resistance induced by pro- inflammatory cytokines may further potentiate hyperglycemia. White adipose tissue represents in addition to its energy storage function also a very active endocrine active organ. In addition to regulation of a number of metabolic processes it also significantly modulates the inflammatory response. In critically ill patients, adipose tissue changes its morphology, i.e. the adipocytes are shrinking and adipose tissue is abundantly infiltrated by macrophages. Paradoxically, overweight and obese critically ill patients have lower mortality than underweight, lean and morbidly obese subjects. In our studies, we selected population of the patients undergoing elective major cardiac surgery with extracorporeal...

Einleitung: Der diätetische Einsatz von ω-3 Fettsäuren wird für viele Erkrankungen sowohl des Menschen als auch der Tiere mit positiven Effekten verbunden. Auch für Tumorerkrankungen wird, insbesondere bei einem niedrigen Gehalt an Vitamin E, eine positive Wirkung von ω-3 Fettsäuren postuliert. Die Aufnahme der ω-3 Fettsäuren beim Hund aus dem Futter sowie die Inkorporation in das Gewebe wird durch viele verschiedene Faktoren beeinflusst. Um den potenziellen therapeutischen Nutzen einer Supplementierung des Futters mit ω-3 Fettsäuren abschätzen zu können, ist es unerlässlich zu wissen, in welchem Ausmaß und in welcher Geschwindigkeit die Inkorporation der ω-3 Fettsäuren aus dem Futter beim Hund erfolgt. Gleichzeitig stellt sich die Frage nach einem geeigneten Indikator, um den Erfolg einer Supplementierung mit ω-3 Fettsäuren zu überprüfen. Zielstellung: In der vorliegenden Dissertation sollten deshalb die folgenden Fragestellungen untersucht werden: a) Kann ein ω-3 Fettsäuresupplement die Fettsäurezusammensetzung im Gewebe genauso effektiv verändern wie ein kommerzielles, ω-3 Fettsäure-reiches Futter? b) Wie gestaltet sich der zeitliche Verlauf der Inkorporation von diätetisch verabreichten ω-3 Fettsäuren in der Erythrozytenmembran (EM) und im Plasma? c) Können die im Plasma zu beobachtenden Veränderungen als Indikator für die Veränderungen in der EM dienen? Material & Methoden: 30 Beagle wurden in 3 Gruppen à 10 Tiere eingeteilt und für 12 Wochen unterschiedlich gefüttert. Die Kontrollgruppe (CONT) erhielt ein kommerzielles Futter, das wenig ω-3 Fettsäuren enthält, eine Versuchsgruppe bekam zusätzlich ein ω-3 Fettsäuren-Konzentrat (ADD) und die zweite Versuchsgruppe erhielt ein kommerzielles Futter mit einem hohen Anteil an ω-3 Fettsäuren (FO). Anschließend wurde ADD für weitere 4 Wochen wie CONT gefüttert. Die Fettsäurezusammensetzung der EM und des Plasmas wurde nach 0, 1, 2, 4, 8 und 12 Wochen und bei ADD zusätzlich auch nach 14 und 16 Wochen per Gaschromatografie analysiert. Der Vitamin E-Gehalt des Plasmas in ADD und CONT wurde per Hochleistungsflüssigkeitsdruckchromatografie bestimmt. Ergebnisse: In unserer Studie erwies sich der Zusatz eines ω-3 Fettsäure-Supplementes zu einem Grundfutter ohne EPA und DHA genau so effektiv wie eine komplette Futterumstellung auf ein kommerzielles ω-3 fettsäurereiches Futter. Dies ist insbesondere für die Therapie von Hunden, die ein Spezialfutter erhalten ein wichtiger Vorteil. Auch können Supplemente einfacher an den jeweiligen individuellen Bedarf angepasst und dosiert werden. Bereits nach einer Woche konnte bei ADD und FO ein signifikanter Anstieg der Gesamt ω-3 Fettsäuren, EPA, und DHA in der EM und im Plasma beobachtet werden. Innerhalb von zwei (ADD) bzw. vier (FO) Wochen war das Plateau des Anstieges der ω-3 Fettsäuren im Plasma erreicht, nach acht Wochen auch in der EM. Das Plateau für DHA wurde im Plasma nach zwei (FO) bzw. vier (ADD) Wochen erreicht, in der EM nach acht Wochen. Das Plateau für EPA wurde im Plasma nach zwei Wochen erreicht, in der EM nach zwei (ADD), bzw. vier (FO) Wochen. Nach der Umstellung von ADD auf CONT-Fütterung ging der Gehalt an EPA im Plasma innerhalb von zwei Wochen auf den Ausgangswert zurück. Der Gehalt an EPA in der EM und der Gehalt an DHA im Plasma und in der EM erreichte das Ausgangsniveau innerhalb der vier Wochen der Washoutperiode nicht wieder. Der Gehalt an Arachidonsäure (AA) und der gesamt ω-6 Fettsäuren in den EM und im Plasma in ADD und FO sank innerhalb des Versuchszeitraumes signifikant, jedoch war die Reduktion nach 12 Wochen noch nicht abgeschlossen. Die Vitamin E Konzentration in ADD und CONT im Plasma zeigte keine signifikanten Änderungen. Schlussfolgerungen: Auf Grund von möglichen individuellen Unterschieden im Fettsäuremuster sollte der Erfolg einer Supplementierung mit ω-3 Fettsäuren immer in Relation zum individuellen Ausgangswert bewertet werden. Unabhängig von der Art der Supplementierung ist ein signifikanter Anstieg von EPA, DHA und der gesamt ω-3 Fettsäuren innerhalb von einer Woche zu erwarten. Hierbei korreliert die Entwicklung im Plasma sehr gut mit der der EM. Die Reduktion von AA und der gesamt ω-6 Fettsäuren erfolgt dagegen über einen wesentlich längeren Zeitraum. Um diese beobachten zu können, ist die Analyse der EM zu bevorzugen.:Abkürzungsverzeichnis ..................................................................................... III 1. Einleitung ....................................................................................................... 1 2. Literatur .......................................................................................................... 3 2.1. Fettsäuren ................................................................................................... 3 2.1.1. Aufbau und Eigenschaften ....................................................................... 3 2.1.2. Vorkommen und Synthese ....................................................................... 4 2.1.3. Funktion der Fettsäuren im Körper .......................................................... 5 2.1.4. Rolle der ω-3 Fettsäuren bei entzündlichen Prozessen .......................... 6 2.2. Tumorerkrankungen .................................................................................... 7 2.2.1 Rolle der ω-3 Fettsäuren bei Tumorerkrankungen .................................... 8 2.3 Rolle der ω-3 Fettsäuren bei anderen Erkrankungen ................................. 12 2.4. Diätetische Versorgung mit ω-3 Fettsäuren ............................................... 12 2.4.1. Inkorporation der ω-3 Fettsäuren in das Gewebe ................................... 13 2.4.2. Indikatoren für den Fettsäurestatus des Organismus .............................. 15 2.5. Vitamin E ..................................................................................................... 16 2.5.1. Aufnahme in den Körper ........................................................................... 16 2.5.2. Funktion von Vitamin E ............................................................................. 17 2.5.3. Hypovitaminose E ..................................................................................... 18 2.5.4. Hypervitaminose E .................................................................................... 18 2.5.5. Supplementierung mit Vitamin E bei Erkrankungen .................................. 18 3. Fragestellungen ............................................................................................... 19 4. Publikationen .................................................................................................... 20 4.1. Publikation 1 .................................................................................................. 20 Stellungnahme zum Eigenanteil der Arbeit an der Publikation 1 .......................... 20 4.2. Publikation 2 .................................................................................................. 32 Stellungnahme zum Eigenanteil der Arbeit an der Publikation 2 .......................... 32 5. Diskussion ......................................................................................................... 43 5.1. Würdigung der Versuchsanstellung ................................................................ 43 5.2. Ausgangssituation ........................................................................................... 45 5.3. Inkorporation der ω-3 Fettsäuren .................................................................... 47 5.4. Auswirkungen auf ω-6 Fettsäuren ................................................................... 49 5.5. Nachteile einer Supplementierung mit ω-3 Fettsäuren ................................... 50 5.6. Effektivität des ω-3 Fettsäure-Additivs ............................................................ 51 5.7. Einsatz von ω-3 Fettsäuren bei Tumorpatienten ............................................. 54 5.8. Einfluss von Vitamin E ..................................................................................... 56 5.9. Indikatoren für den Erfolg einer Supplementierung mit ω-3 Fettsäuren .......... 58 5.10. Ausblick ......................................................................................................... 59 6. Schlussfolgerungen ............................................................................................ 60 7. Zusammenfassung ............................................................................................. 61 8. Summary ............................................................................................................ 63 9. Literaturverzeichnis ............................................................................................ 65 Danksagung ........................................................................................................... 83 / Introduction: Dietary supplementation with n-3 fatty acids is associated with positive effects on many diseases in humans and animals. A positive effect of n-3 fatty acids on cancer is discussed especially in combination with a low Vitamin E content. Bioavailability from food and incorporation of n-3 fatty acids into tissues is influenced by many different factors. In order to estimate the potential therapeutical use of n-3 fatty acid supplementation in dogs it is nessecary to know the extend and speed of the incorporation of dietary n-3 fatty acids into tissues. There is also need for a reliable indicator to monitor the success of n-3 fatty acid supplementation. Objective: We therefore sought to answer the following questions: a) Is a n-3 fatty acid additive as effective in changing tissue fatty acid profiles as a commercial n-3 fatty acid diet? b) How are n-3 fatty acids incorporated into erythrocyte membranes (EM) and plasma over time? c) Are plasma fatty acid profiles a suitable indicator for EM fatty acid profiles? Material & Methods: 30 Beagle dogs were divided into 3 groups with 10 dogs per group and fed different diets for 12 weeks. One group got a commercial diet with a low n-3 fatty acid content (CONT). One group got the CONT diet with an added n-3 fatty acid concentrate (ADD) and one group got a commercial diet rich in n-3 fatty acids. After the 12 week period ADD was fed an additional four weeks as CONT to observe washout effects. Fatty acid profiles of plasma and EM were analysed at week 0, 1, 2, 4, 8 and 12 and for ADD also at week 14 and 16 per gas chromatography. Vitamin E content was analysed in Plasma of ADD and CONT via high pressure liquid chromatography. Results: In our study the use of a n-3 fatty acid additive was as effective in changing tissue fatty acid profiles as a commercial diet rich in n-3 fatty acids. Especially for dogs already recieving specialized diets, this is an important advantage. Additives are also much easier to customise and dose according to individual needs. A significant increase of total n-3 fatty acids, EPA and DHA was seen in EM and in plasma after one week both in ADD and FO. For total n-3 fatty acids the plateau was reached in plasma after two (ADD) and four (FO) weeks and after eight weeks in EM. DHA reached its plateau in plasma after two (FO) and four (ADD) weeks and after eight weeks in EM. For EPA the plateau was reached after two weeks in plasma and in EM after two (ADD) and four (FO) weeks. During the washout period in ADD EPA reached its baseline levels after two weeks in plasma but not within four weeks in EM. Total n-3 fatty acids and DHA in both plasma and EM also did not return to baseline levels within the four weeks of the washout period. Arachidonic acid (AA) and total n-6 fatty acids were significantly reduced in both ADD and FO during the trial, but their decline was not completed within the 12 weeks of the trial period. Vitamin E content in ADD and CONT showed no significant changes. Conclusion: Due to possible individual differences in fatty acid profiles success of dietary n-3 fatty acid supplementation should always be measured in relation to individual fatty acid profiles before the start of dietary supplementation. Both the additive and the commercial n-3 fatty acid diet led to an increase in EPA, DHA and total n-3 fatty acids within one week. This could be seen clearly in both plasma and EM. Changes in EM also correlated well with changes in plasma. For AA and total n-6 Fatty acids it took much longer to decline. In order to monitor this decline analysis of EM should be preferred.:Abkürzungsverzeichnis ..................................................................................... III 1. Einleitung ....................................................................................................... 1 2. Literatur .......................................................................................................... 3 2.1. Fettsäuren ................................................................................................... 3 2.1.1. Aufbau und Eigenschaften ....................................................................... 3 2.1.2. Vorkommen und Synthese ....................................................................... 4 2.1.3. Funktion der Fettsäuren im Körper .......................................................... 5 2.1.4. Rolle der ω-3 Fettsäuren bei entzündlichen Prozessen .......................... 6 2.2. Tumorerkrankungen .................................................................................... 7 2.2.1 Rolle der ω-3 Fettsäuren bei Tumorerkrankungen .................................... 8 2.3 Rolle der ω-3 Fettsäuren bei anderen Erkrankungen ................................. 12 2.4. Diätetische Versorgung mit ω-3 Fettsäuren ............................................... 12 2.4.1. Inkorporation der ω-3 Fettsäuren in das Gewebe ................................... 13 2.4.2. Indikatoren für den Fettsäurestatus des Organismus .............................. 15 2.5. Vitamin E ..................................................................................................... 16 2.5.1. Aufnahme in den Körper ........................................................................... 16 2.5.2. Funktion von Vitamin E ............................................................................. 17 2.5.3. Hypovitaminose E ..................................................................................... 18 2.5.4. Hypervitaminose E .................................................................................... 18 2.5.5. Supplementierung mit Vitamin E bei Erkrankungen .................................. 18 3. Fragestellungen ............................................................................................... 19 4. Publikationen .................................................................................................... 20 4.1. Publikation 1 .................................................................................................. 20 Stellungnahme zum Eigenanteil der Arbeit an der Publikation 1 .......................... 20 4.2. Publikation 2 .................................................................................................. 32 Stellungnahme zum Eigenanteil der Arbeit an der Publikation 2 .......................... 32 5. Diskussion ......................................................................................................... 43 5.1. Würdigung der Versuchsanstellung ................................................................ 43 5.2. Ausgangssituation ........................................................................................... 45 5.3. Inkorporation der ω-3 Fettsäuren .................................................................... 47 5.4. Auswirkungen auf ω-6 Fettsäuren ................................................................... 49 5.5. Nachteile einer Supplementierung mit ω-3 Fettsäuren ................................... 50 5.6. Effektivität des ω-3 Fettsäure-Additivs ............................................................ 51 5.7. Einsatz von ω-3 Fettsäuren bei Tumorpatienten ............................................. 54 5.8. Einfluss von Vitamin E ..................................................................................... 56 5.9. Indikatoren für den Erfolg einer Supplementierung mit ω-3 Fettsäuren .......... 58 5.10. Ausblick ......................................................................................................... 59 6. Schlussfolgerungen ............................................................................................ 60 7. Zusammenfassung ............................................................................................. 61 8. Summary ............................................................................................................ 63 9. Literaturverzeichnis ............................................................................................ 65 Danksagung ........................................................................................................... 83

Bioluminescence is the chemical production of light that results when a luciferase enzyme catalyzes the luminogenic oxidation of a small-molecule luciferin substrate. The numerous luciferases and luciferins nature has evolved can be used to illuminate biological processes, from in vitro assays to imaging processes in live animals. However, we can improve the utility of bioluminescence through modification of these enzymes and substrates. My thesis work focuses on developing reporters that expand the bioluminescent toolkit and improving our understanding of how bioluminescence works on a molecular level. The first part of my thesis focuses on characterizing luciferases and luciferins that improve bioluminescence imaging in vivo. Some of our luciferins can outperform the natural D-luciferin substrate in live mouse imaging, while others are selectively utilized by mutant luciferases in live mouse brain. We also engineered luciferins that can selectively report on endogenous enzymatic activity in live mice. The second part of my thesis focuses on determining the molecular details of how enzymes related to firefly luciferase, long-chain fatty acyl-CoA synthetases (ACSLs), can function as latent luciferases. I have determined the structure for one of these enzymes and improved its bioluminescent activity with synthetic luciferins enough to image in live mouse brain. I also characterized the selectivity in chimerized enzymes that combine firefly luciferase and ACSLs. In summary, my work improves the utility of bioluminescence for in vivo use and informs us about how evolutionarily-related enzymes function as luciferases on a molecular level.

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / It has been well documented that omega-3 PUFA (n-3 PUFA) can confer a wide variety of health benefits to humans and animals. The current study was designed to evaluate the ability of n-3 PUFA to modulate the innate immune response in two diverse breeds of horses. Ten Quarter Horse and 10 American Miniature Horse mares were assigned to either an n-3 PUFA supplemented or control diet (5 full-sized and 5 miniature mares/treatment) for 56 d. The treatment diet was designed to deliver 64.4 mg/kg BW combined eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) daily. Whole blood (20 mL) was collected via jugular veinipuncture into heparinized tubes on 0 d, 28 d, and 56 d. Serum PUFA analysis was conducted by gas chromatography. Peripheral blood mononuclear cell (PBMC) production of tumor necrosis factor-alpha (TNFalpha) in response to toll-like receptor (TLR) ligands lipopolysaccharide (LPS), flagellin, and lipoteichoic acid (LTA) was estimated using an equine-specific ELISA. Peripheral blood samples from d 56 were also analyzed for total and differential leukocyte counts and subjected to flow cytometric analysis. Body type did not affect basal or TLR stimulated TNFα production. Serum PUFA analysis revealed a decrease in linoleic acid (LA) and substantial increases in arachidonic acid (ARA), EPA, DHA, and docosapentaenoic acid (DPA) at both d 28 and 56 in horses fed n-3 PUFA (P less than 0.0001 for all). Dietary n-3 PUFA supplementation reduced (P less than 0.05) un-stimulated basal, but not TLR stimulated TNFalpha production by PBMC’s. Supplementation with n-3 PUFA did not affect total or differential leukocyte counts, nor selected cell surface markers. These results suggest that n-3 PUFA supplementation in the horse can modify circulating PUFA and alter the inflammatory response by reducing basal TNFalpha production. Furthermore, under conditions of the current study and considering the endpoints evaluated, the American Miniature Horse could potentially be used as a model for full-sized horse breeds.

BACKGROUND:Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.RESULTS:A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.CONCLUSIONS:The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

The main objective of this thesis was to gain new insights in three of Florida’s high value food and stock enhancement finfish nutrition (Common snook, Florida pompano and red drum) to improve larviculture protocols. The main bottleneck in snook production is the extremely low larval survival rate, which hinders subsequent research. This work first focused on the source of the larvae by looking at potential nutritional deficiencies in captive broodstock. The lipid composition of wild and captive common snook broodstock were compared to identify disparities and gain the information necessary for the formulation of a suitable diet for captive stocks. Results showed that captive snook lipid content was significantly higher than that of wild fish. However, cholesterol and arachidonic acid (ARA) levels were significantly lower compared to wild broodstock, with potential impact on steroid and prostaglandin production, reproductive behavior and gametogenesis. Eggs from captive broodstock incorporated high docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) levels and low ARA levels. Consequently, ARA/EPA ratio in captive eggs was more than half of that in wild eggs (2.3 ± 0.6 and 0.9 ± 0.1 respectively), with a probable perturbation in eicosanoid production and adverse effects on embryo and larval development. The large differences observed between wild and captive broodstock most likely contributed to the reproductive dysfunctions observed in captive snook broodstock (e.g. incomplete oocyte maturation, low milt production and poor egg quality). In addition, the presence of hydrocarbons was detected in the liver of most of the wild snook sampled. This requires further investigation to identify the source of the contamination, monitor a potential impact on reproductive performances and protect the species habitat. Another major bottleneck in marine fish rearing occurs during the transition from endogenous feeding to exogenous feeding, with mass mortality events linked to inadequate first feeding diets. To gain insight on the early fatty acid requirements and mobilization of pompano and snook larvae, the pattern of conservation and loss of fatty acids from the yolk sac during the endogenous feeding period and subsequent starvation was studied. In both species, fatty acids were utilized as an energy source after hatching. Mono-unsaturated fatty acids were catabolized, while saturated and poly-unsaturated fatty acids were conserved. High levels of arachidonic acid (ARA) in pompano and snook eggs (respectively 2.44 ± 0.1 and 5.43 ± 0.3 % of total fatty acids), as well as selective retention in the unfed larvae, suggested a high dietary requirement for this fatty acid during the early stages of larval development. The effect of an ARA supplementation was therefore investigated in snook larvae at the rotifer feeding stage. Larvae receiving the supplementation did incorporate higher levels of ARA, and DHA/EPA and ARA/EPA ratios were successfully modified to match those observed in wild eggs. No significant improvements in growth or survival were observed, however the success in fatty acid profile modification suggest a possible impact of the supplementation on a longer period of time and a possible effect on stress resistance. Probiotics have been shown to enhance larval performances of several species and this strategy was therefore investigated to evaluate a potential impact on Florida pompano, red drum and common snook larvae. The effect of a commercial mix of Bacillus sp. was studied on larval survival, growth and digestive enzyme activities. Larvae were fed either live feed enriched with Algamac 3050 (Control), Algamac 3050 and probiotics (PB), or the previous diet combined with a daily addition of probiotics to the tank water (PB+). Microbiological analyses were performed at the end of the pompano trial. Numbers of presumptive Vibrio sp. were low and not statistically different between treatments, therefore no additional microbiological analyses were performed on the system. At the end of the pompano and snook trial, standard lengths of larvae from the PB and PB+ treatments were significantly greater than for the control larvae. For both pompano and snook, trypsin specific activity was higher in PB and PB+ larvae compared to the control larvae. Similarly, alkaline phosphatase activity was higher for the pompano larvae fed the PB and PB+ treatments and for the snook larvae fed the PB+ treatment compared to the control larvae. No enhancement of growth or digestive enzymes activities was observed in red drum larvae. Yet, no negative effects were noticed and a longer trial period and the study of additional parameters could reveal different effects. In all three species, survival was not affected by the supplementation; however, stress exposure should be further investigated as the supplementation may strengthen the larvae, especially pompano and snook larvae where the Bacillus sp. supplementation appears to promote growth through an early maturation of the digestive system. Another key challenge in marine fish larval rearing resides in weaning the larvae onto dry micro-diets. This step is commonly concurrent with larvae metamorphosis into juveniles, with extensive morphological and physiological changes that are likely to influence nutritional requirements. In the present project, three microdiets were tested on weaning of Florida pompano larvae: Otohime, Gemma and a reference diet LR803. The experimental system was stocked with 11-day-old larvae, which were co-fed micro-diets and live food from 11 dph to 17 dph then micro-diets only until 28 dph. Survival from 11 dph to 28 dph was similar for all treatments, with an average of 33 %. At the end of the trial, the Gemma larvae were significantly longer and heavier than larvae fed the other diets. Fatty acid composition of the diets and larvae varied significantly between treatments. The Gemma larvae incorporated the lowest amount of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA). However, they presented the highest DHA/EPA and ARA/EPA ratios, supporting the concept that the proportions of polyunsaturated fatty acids are of greater importance than their absolute amount. Results from the enzyme analysis showed that fishmeal is a suitable main source of protein for Florida pompano larvae and demonstrated the full functionality of the pancreas at 16 days post hatch. These results provide the basis of a suitable weaning diet for pompano larvae and indicate the possibility of a weaning time prior to 16 days post hatch, which is of high interest in commercial production. Overall, this research provides new data on common snook, pompano and red drum nutritional requirements with results that can be directly applied to help overcome major bottlenecks in the hatchery phase and improve rearing protocols.

<p>U okviru doktorske disertacije ispitan je nutritivni kvalitet mleka magarice balkanske rase tokom laktacije. Prosečna suva materija mleka magarice balkanske rase iznosi 9,26%. Sadržaj proteina tokom laktacije kreće se od 1,40% do 1,92%. Prosečan sadržaj mlečne masti je 0,61%, a sadržaj laktoze iznosi 6,50%. Sadržaj analiziranih minerala: Ca, Na, K, Mg, P i Zn se povećava tokom laktacije i maksimalna vrednost utvrđena je 170. dana. Primenom kapilarne elektroforeze definisan je profil proteina mleka magarice balkanske rase. Identifikovane su sledeće proteinske frakcije: &alpha;s1-kazein, &alpha;s2-kazein, &beta;-kazein (A, F), &alpha;-laktalbumin (A, C), &beta;-laktoglobulin, lizozim, laktoferin, serum albumin i imunoglobulin čiji sadržaj opada tokom perioda laktacije. Sadržaj &alpha;-laktalbumina se kreće od 3090 mg/l do 1990 mg/l, a lizozima varira od 1040 mg/l do 2970 mg/l. Navedene frakcije proteina su najzastupljenije u mleku magarice balkanske rase. Laktoferin i imunoglobulin su frakcije sa najmanjim udelom u mleku magarice balkanske rase. Kori&scaron;ćenjem gasne hromatografije/masene spektrometrije utvrđen je sastav masnih kiselina mleka. Udeo esencijalne linolne kiseline (C18:2 n6) kreće se u opsegu od 7,08%, do 9,69%, a udeo &alpha;-linoleinske kiseline (C18:3 n3) varira od 5,85% do 7,83%.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sastav mleka magarice balkanske rase kompariran je sa nutritivnim karakteristikama humanog mleka tokom 40. i 90. dana laktacije. Utvrđene su značajne razlike u sadržaju proteina mleka, mlečne masti i minerala. Odnos kazeina i proteina surutke kreće se od 0,68 do 0,75 u mleku magarice, dok u humanom mleku varira od 0,59 do 0,70. Udeo -linoleinske kiseline (C18:3 n3) je oko 2,5 puta veći u mleku magarice u odnosu na humano mleko.<br />Generalno se može zaključiti da mleko magarice balkanske rase ima specifične nutritivne karakteristike koje variraju u zavisnosti od sastava hrane za životinje i analiziranog perioda laktacije.</p> / <p>Nutritional quality of Balkan donkey milk during lactation was investigated within this thesis. The mean content of dry matter, fat and lactose in the Balkan donkey milk was 9.26%, 0.61% and 6.50%, respectively. Protein content during lactation period ranged from 1.40% to 1.92%. Content of the analyzed minerals: Ca, Na, K, Mg, Zn and P increased during the lactation period and reached their maximum value at 170th day. The protein profile of Balkans donkey milk was defined by application of capillary electrophoresis when the following protein fractions: &alpha;s1-kazein, &alpha;s2-kazein, &beta;-kazein (A, F), &alpha;-laktalbumin (A, C), &beta;-laktoglobulin, lysozyme, lactoferrin, serum albumins and immunoglobulins, whose content decreases during lactation period,were identified. &alpha;-lactalbumin contents ranged from 3090 mg/l to 1990 mg/ and lysozyme varies between 1040 mg/l to 2970 mg/l. These two protein fractions were the most abundant in the Balkan donkey milk, while lactoferrin and immunoglobulin were at least represented. The fatty acid composition of Balkan donkey milk was determined using gas chromatography/mass spectrometry. The share of the essential linoleic (C18: 2 n6) and &alpha;&nbsp;- linolenic (C18: 3 n3) acid rangred from 7.08 % to 9.69% and from 5.85 % to 7.83 %.</p><p>Nutritional quality of Balkan donkey milk has been compared with the nutritional quality of human milk during the 40th and 90th day of lactation. Significant differences in the protein content of milk, fat and minerals were found. The ratio of casein and whey protein ranged from 0.68 to 0.75 in the Balkan donkey milk, while in human milk this value varies from 0.59 to 0.70. The share of &alpha;-linolenic acid (C18:3 n3) is around 2.5 times higher in donkey than in human milk.<br />The main conclusion is that Balkan donkey milk has specific and unique nutritional quality which depend on the feed composition and on the analyzed period of lactation.</p>

Biodiesel use and production has significantly increased in the United States and in other parts of the world in the past decade. This change is driven by energy security and global climate legislation mandating reductions in the use of petroleum-based diesel. Recent air quality research has shown that emission of some pollutants such as CO, particulate matter (PM), SO2, hydrocarbons, and carcinogenic polycyclic aromatic hydrocarbons (PAHs) is greatly reduced with biodiesel. However, studies have also shown that some unregulated emissions, such as gas-phase carbonyls, are increased with biodiesel combustion. Very limited research has been done to investigate the particle-phase carbonyl and quinone emissions from biodiesel combustion. Also, very limited studies have investigated the ozone oxidation of biodiesel exhaust PM. Fatty acid methyl esters (FAMEs) are found in high abundance in biodiesel exhaust PM. The presence of these FAMEs in biodiesel exhaust PM can potentially alter the kinetics of the reactions between ozone and particle-phase PAHs. In this study, an Armfield CM-12 automotive light-duty diesel engine operated on a transient drive cycle was used to generate PM from various waste vegetable oil (WVO) and soybean biodiesel blends (containing 0%, (B00), 10% (B10), 20% (B20), 50% (B50), and 100% (B100) biodiesel by volume). The primary PM emissions were sampled using Teflon-coated fiberfilm filters. Laboratory PAHs, FAMEs, and B20 exhaust PM were exposed to ~0.4 ppm ozone for time periods ranging from 0-24 hours in order to study the effect of FAMEs and biodiesel exhaust PM on the ozonolysis of PAHs. Organic chemical analysis of samples was performed using gas chromatography/mass spectrometry (GC/MS). PAHs, carbonyls, FAMEs, and n-alkanes were quantified in the exhaust PM of petrodiesel, WVO and soybean fuel blends. The emission rates of the total PAHs in B10, B20, B50, and B100 exhaust PM decreased by 0.006-0.071 ng/µg (5-51%) compared to B00, while the emission rates for the FAMEs increased with increasing biodiesel content in the fuel. The emission rates of the total n-alkanes in B10, B20, B50, and B100 exhaust PM decreased by 0.5-21.3 ng/µg (4-86%) compared to B00 exhaust PM. The total emission rates of the aliphatic aldehydes in biodiesel exhaust PM (B10, B20, B50, and B100) increased by 0.019-2.485 ng/µg (36-4800%) compared to petrodiesel. The emission rates of the total aromatic aldehydes, total aromatic ketones, and total quinones all generally decreased with increasing biodiesel content in the fuel. With the exception of benzo[a]pyrene, the pseudo-first order ozone reaction rate constants of all the PAHs decreased by 1.2-8 times in the presence of the FAMEs. Phenanthrene, fluoranthene, and pyrene were the only PAHs detected in the B20 exhaust PM, and their ozone reaction rate constants were about 4 times lower than those obtained when the PAHs alone were exposed to ozone. The findings of this study indicate that there are both positive and negative effects to emissions associated with biodiesel use in light-duty diesel engines operating on transient drive cycle.

Chez la plante modèle Arabidopsis, le processus de maturation de la graine et, en particulier, l’accumulation de composés de réserves (huile et protéines de réserve) sont étudiés depuis de nombreuses années. Si les voies de biosynthèse conduisant à l’accumulation de tels composés sont bien décrites, leur régulation est encore largement méconnue. Mon travail de thèse s’inscrit dans un projet de recherche dont le but est d’identifier de nouveaux régulateurs transcriptionnels de la maturation de la graine d’Arabidopsis. Après avoir réalisé une étude comparative du processus de maturation chez les deux zygotes de la graine, embryon et albumen, nous avons caractérisé un facteur de transcription appelé MYB118, exprimé spécifiquement dans l’albumen et potentiellement impliqué dans la régulation du processus de maturation. Son patron d’expression, finement caractérisé, montre un pic d’accumulation d’ARNm en début de maturation de la graine, plus spécifiquement dans l’albumen. Des études menées sur des lignées mutantes ou surexprimant LEAFY COTYELDON2 (LEC2) révèlent que l’expression de MYB118 est activée par ce régulateur maître de la maturation de la graine. Une analyse biochimique de graines myb118 montre que le contenu en huile et en protéines de réserve est doublé dans l’albumen et réduit dans l’embryon de ce mutant par comparaison aux graines sauvages. Finalement, une analyse transcriptomique effectuée sur des graines myb118 a permis d’identifier des cibles putatives dont la dérégulation pourrait expliquer le phénotype : MYB118 semble être un répresseur de l’accumulation de composés de réserve dans l’albumen. Comme la famille de facteurs de transcription à laquelle appartient MYB118 comprend de nombreux membres, nous nous sommes intéressés au patron d’expression et au rôle de ses paralogues les plus proches. L’un d’entre eux, appelé MYB115, est exprimé spécifiquement dans l’albumen chalazal et semble avoir une fonction partiellement redondante à celle de MYB118. / In the model plant Arabidopsis thaliana, seed maturation and more especially the accumulation of storage compounds such as oil and seed storage proteins (SSP) have been widely studied. Although the biosynthetic networks underlying the accumulation of such compounds are now well described, regulation of these pathways remains poorly understood. My Ph.D. project is a part of a research program aimed at identifying new transcriptional regulators of seed maturation in Arabidopsis. After a comparative analysis of maturation processes in the two zygotic tissues of the seed, namely the embryo and the endosperm, we have characterized MYB118, an endosperm-specific transcription factor, putatively involved in the regulation of this maturation process. A fine and comprehensive characterization of its expression pattern showed a peak of expression at the onset of the maturation phase in the endosperm. Expression studies carried out in LEAFY COTYLEDON2 (LEC2) mutant and over-expressing lines demonstrated that MYB118 expression is positively regulated by this master regulator. Biochemical analysis of myb118 seeds showed that oil and SSP contents were doubled in the endosperm fraction and decreased in the embryo of this mutant compared to the wild type. A transcriptomic analysis of myb118 mutant seeds point out some putative targets, the misregulation of which could explain this phenotype: MYB118 seems to be a repressor of storage compounds accumulation in the endosperm.Since MYB118 belongs to the broad family of MYB transcription factors, we investigated the expression pattern and the role of its closest paralogs. One of them, called MYB115 is expressed specifically in the chalazal endosperm and seems to have partially redundant functions with MYB118.

La lipémie postprandiale se caractérise par une augmentation des lipoprotéines riches en triglycérides après un repas, et joue un rôle important dans la biodisponibilité des lipides alimentaires pour les tissus périphériques. En effet, une lipémie postprandiale élevée est souvent associée à l'obésité et à une dyslipidémie, deux composantes du syndrome métabolique qui peuvent engendrer des complications médicales, incluant diabète et maladies cardiovasculaires. La lactoferrine (Lf) inhibe l'épuration hépatique des chylomicrons, conduisant à une élévation de la lipémie postprandiale par des mécanismes moléculaires non élucidés. Il est aussi établi que le Lipolysis Stimulated Receptor (LSR) contribuait à l'épuration des lipoprotéines riches en triglycérides pendant la phase postprandiale. L'objectif était de déterminer s'il existait une interaction entre la Lf et le LSR. Les études de cultures cellulaires ont montré que si la Lf n'affectait pas le taux d'expression du LSR dans des cellules Hepa 1-6 de souris, elle co-localisait avec le LSR en présence d'oléate, un composé requis pour l'activation du récepteur. Des expériences de ligand-blotting ont également montré que la Lf se fixait sur le LSR purifié et inhibait la fixation de lipoprotéines riches en triglycérides. Les domaines N et C-terminaux isolés de cette protéine, ainsi qu'un mélange de peptides obtenu après double hydrolyse de la Lf par la trypsine et la chymotrypsine, conservent cette propriété. Nous proposons que l'élévation de la lipémie postprandiale observée in vivo suite à un traitement par la Lf soit médiée par son interaction avec le LSR, inhibant ainsi l'épuration des chylomicrons et de leurs remnants / Postprandial lipemia is characterized by an increase in plasma triglyceride-rich lipoproteins after the ingestion of meal, and is important towards determining the bioavailability of dietary lipids amongst the peripheral tissues. Indeed, elevated postprandial lipemia is often observed with obesity and dyslipidemia, two disorders that can lead to health complications including diabetes and cardiovascular diseases. Lactoferrin (Lf), has been shown to inhibit hepatic chylomicron remnant removal, resulting in increased postprandial lipemia, for which the molecular mechanisms remain unclear. The lipolysis stimulated lipoprotein receptor (LSR) has been shown to contribute to the removal of triglycerides-rich lipoproteins during the postprandial phase. The aim was to determine if there was interaction between Lf and LSR. Both Lf and LSR were purified with purities upper to 95% and characterized. Cell culture studies demonstrated that while Lf does not have any significant effect on LSR protein levels in mouse Hepa1-6 cells, it co-localizes with LSR in cells, but only in the presence of oleate, which is needed to obtain LSR in its active form. Ligand blotting using purified LSR revealed that Lf binds directly to the receptor in the presence of oleate and prevents the binding of triglycerides-rich lipoproteins. Both C- and N-lobes of Lf, and a mixture of peptides derived from its tryptic and chymotryptic double hydrolysis retained the ability to bind LSR. We propose that the elevated postprandial lipemia observed upon Lf treatment in vivo is mediated by its direct interaction with LSR, thus preventing clearance of chylomicrons and their remnants through the LSR pathway