The biosynthesis of wax components containing secondary functional groups was investigated in the current study. Two fundamentally different pathways were proposed to introduce the secondary functional groups. One pathway involves hydroxylation of elongated substrates. Wax components characterized by two functional groups located on or near the centre of the carbon chain, nonacosane-14,15-diol, -14,16-diol and -13,15-diol as well as corresponding ketols were identified for the first time in Arabidopsis stem wax. The alkanediols and ketols were dominated by the C-14,15 isomers. The absence of alkanediols and ketols in Arabidopsis mah1 mutants that are deficient in secondary alcohol biosynthesis confirmed the biosynthetic relationship between secondary alcohols and alkanediols/ketols (Chapter 3). In pea (Pisum sativum) leaf wax, two novel compound classes were identified as primary/secondary alcohols dominated by octacosane-1,14-diol and secondary/secondary alkanediols hentriacontane-9,16-diol, -8,15-diol and -10,17-diol. Co-localization of the secondary/secondary alkanediols and hentriacontan-15-ol and -16-ol pointed to a biosynthetic relationship (Chapter 4). The diverse structures of compounds identified in the current study suggested that hydroxylases can use substrates other than alkanes. The predominance of isomers within homologues indicated a regiospecificity of the hydroxylases involved in wax biosynthesis. In addition to hydroxylation, secondary functional groups could also be introduced through elongation of carbon chains. Homologous series of 5-hydroxyaldehydes (C₂₄ and C₂₆-C₃₆) and 1,5-alkanediols (C₂₈-C₃₈) were identified in yew (Taxus baccata) needle wax. The relative position of both functional groups suggested that these two compound classes are biosynthetically related and their secondary functional groups are introduced during elongation (Chapter 5). The results of incubation of ¹⁴C-labeled malonyl-CoA and acyl-CoAs with different chain lengths in the presence of California poppy (Eschscholzia californica) microsomes provided the first evidence to support the elongation hypothesis. The results indicated that a carbonyl group rather than a hydroxyl group is introduced during elongation. To provide molecular tools for further investigations of the hypothetical pathway, three full length cDNAs encoding putative KCSs were cloned and one of them, PKCSI, was functionally characterized.

Els residus carnis, o subproductes animals, són interessants per al procés de digestióanaeròbia i producció de biogàs, donat el seu elevat potencial energètic i l'actual marc legislatiu que prima la producció d'energia renovable. Tot i així, l'elevat contingut en lípids i proteïnes d'aquests residus pot limitar el seu tractament en introduir fenòmens d'inhibició, dels quals el més important és el produït pels àcids grassos de cadena llarga (AGCL), resultants de la hidròlisi dels lípids. L'objectiu de la present tesis és aprofundir en el coneixement d'aquest procés d'inhibició, en la capacitat d'adaptació dels microorganismes i en la recuperació o prevenció dels fenòmens d'inhibició. En una primera aproximació a la problemàtica, es caracteritzen residus orgànics d'escorxador, s'estudia la seva biodegradabilitat anaeròbia amb diferents relacions lípids/proteïnes i es realitzen assaigs discontinus seqüencials incrementant la concentració de substrat mitjançant pulsos consecutius. Es comprova que la hidròlisi i acidogènesi de proteïnes és molt ràpida i que la degradació dels lípids i AGCL limita la velocitat global del procés. Malgrat aquesta limitació, el sistema es recupera després dels pulsos aplicats, tot augmentant la taxa màxima de producció de metà. Per tal d'estudiar el fenòmen de recuperació, s'estudien i desenvolupen diferents estratègies en reactors sotmesos a processos d'inhibició per AGCL. L'increment dels ratis biomassa/AGCL o l'adició d'additius com la bentonita, per tal de reduir la biodisponibilitat o l'adsorció dels AGCL sobre la biomassa activa, es mostren com estratègies funcionals d'utilitat en l'operació de plantes industrials. Els resultats obtinguts reforcen la hipòtesi de que la inhibició és deguda a adsorció d'AGCL sobre la membrana cel·lular i que la recuperació es pot mesurar mitjançant un augment de l'activitat dels microorganismes. Per tal de dilucidar sobre la natura del augment de l'activitat en els processos de recuperació es caracteritza la inhibició-recuperació mitjançant tres tècniques: 1) estudi de les activitats dels microorganismes a diferents substrats 2) utilització de tècniques de biologia molecular per caracteritzar les poblacions, i 3) desenvolupant expressions cinètiques del procés d'inhibició, basades en l'adsorció, en el marc del model matemàtic ADM1 de la International Water Association. Mitjançant aquestes metodologies es comprova que els fenòmens d'inhibició i adaptació es poden explicar mitjançant un creixement poblacional específic i la inclusió dels fenòmens físic d'adsorció en el procés d'inhibició metabòlica. Finalment, s'avalua de forma més detallada el procés d'adsorció-inhibició mitjançant la determinació de les isotermes d'adsorció i monitoritzant mitjançant assaigs amb biomassa granular i tècniques de microscòpia de fluorescència. Aquesta caracterització ha permès obtenir estratègies de prevenció de la inhibició per AGCL, mitjançant competència amb adsorbents sintètics, i concloure que l'àcid palmític és el limitant en el procés de -oxidaciódels AGCL. Els resultats obtinguts constitueixen una base per al millor coneixement de les possibilitats de tractament anaerobi del residus carnis i dels processos d'inhibició per AGCL i adaptació de la biomassa. El procés físic d'adsorció ha estat directament relacionat amb el fenòmen d'inhibició metabòlica, obtenint-se una descripció matemàtica del mateix. Els resultats han permès plantejar estratègies operacionals, sent una eina a disposició d'operadors de plantes de biogàs per optimitzar la producció d'energia d'aquests residus >mitjançant la seva digestió anaeròbia. / Los residuos cárnicos, o subproductos animales, son interesantes para el proceso de digestión anaerobia y producción de biogás, dado su elevado potencial energético y el actual marco legal que prima la producción de energía renovable. A pesar de esto, el elevado contenido en lípidos y proteínas puede limitar su tratamiento, al introducir fenómenos de inhibición, de los cuales el más importante es el producido por ácidos grasos de cadena larga (AGCL), resultado de la hidrólisis de los lípidos. El objetivo de la presente tesis es profundizar en el conocimiento de este proceso de inhibición, en la capacidad de adaptación de los microorganismos t en la recuperación de sistemas inhibidos. En una primera aproximación a la problemática, se caracterizan los residuos orgánicos de matadero, se estudia su biodegradabilidad anaerobia con diferentes relaciones lípido/proteína y se realizan ensayos discontinuos secuenciales incrementando la concentración de substrato mediante pulsos consecutivos. Se comprueba que la hidrólisis y acidogénesis de las proteínas es muy rápido y que la degradación de lípidos y AGCL limita la velocidad global del proceso. A pesar de esta limitación, el sistema se recupera después de los pulsos aplicados aumentando la tasa máxima de producción de metano. A fin de estudiar el fenómeno de recuperación, se estudian y desarrollan diferentes estrategias en reactores inhibidos por AGCL. El incremento de los ratios biomasa/AGCL o la adición de aditivos como la bentonita, a fin de reducir la biodisponibilidad o la adsorción de los AGCL sobre la biomasa activa, se muestran estrategias funcionales de utilidad en la operación de plantas industriales. Los resultados obtenidos refuerzan la hipótesis de que la inhibición es debida a adsorción de AGCL sobre la membrana celular y que la recuperación se puede medir mediante un aumento de la actividad de los microorganismos. A fin de dilucidar sobre la naturaleza del aumento de la actividad en los procesos de recuperación se caracteriza la inhibición mediante tres técnicas: 1) estudio de las actividades de los microorganismos a diferentes substratos, 2) utilización de técnicas de biología molecular para caracterizar las poblaciones, y 3) desarrollando expresiones cinéticas del proceso de inhibición, basado en la adsorbió, en el marco del modelo ADM1 de la International Water Association. Mediante estas metodologías se comprueba que los fenómenos de inhibición y adaptación se pueden explicar mediante un crecimiento poblacional específico y la inclusión de la adsorción en el proceso de inhibición metabólica. Finalmente, se evalúa de forma detallada el proceso de adsorción-inhibición mediante la determinación de las isotermas de adsorción y monitorizando estos procesos mediante ensayos discontinuos con biomasa granular y técnicas de microscopia de fluorescencia. Esta caracterización ha permitido obtener estrategias de prevención de la inhibición por AGCL, mediante competencia con adsorbentes sintéticos, y concluir que el ácido palmítico es el limitante en el proceso de mutante -oxidación de los AGCL. Los resultados obtenidos constituyen una base para el mejor conocimiento de las posibilidades de tratamiento anaerobio de residuos cárnicos y de los procesos de inhibición por AGCL y adaptación de la biomasa. El proceso físico de adsorción se ha relacionado directamente con el fenómeno de inhibición metabólica, obteniéndose una descripción matemática del mismo. Los resultados han permitido plantear estrategias operacionales, siendo una herramienta a disposición de operadores de plantas de biogás para optimizar la producción de energía de estos residuos mediante su digestión anaerobia. / Slaughterhouse wastes are interesting for the anaerobic digestion process regarding its high biogas production potential and because the current legal scenario promotes renewable energy production. The high lipid and protein content of those residues limit its treatment due to inhibitory processes, in particular the inhibition caused by long chain fatty acids (LCFA). The objective of the present disertation is to obtain a deeper insight on the LCFA inhibition process, the microorganism adaptation ability and the prevention/recovery of inhibitory phenomena. In a preliminary approach, organic wastes generated in slaughterhouses are characterized, by studying the anaerobic biodegradability of waste mixtures containing diferents lipid/proteins concentrations. Anaerobic batch tests are performed at increasing substrate concentrations by sequential pulse feeding. From those experiments, the fast hydrolysis-acidogenesis of proteins is verified, being the lipids and LCFA degradation the main limiting step of the overall anaerobic process. Despite this limitation, the system is able to recover up to a higher methane production rate after each applied pulse. In order to elucidate on the mechanisms of the recovery process, several strategies to recover LCFA inhibited reactors are tested. The increase of the biomass/LCFA ratio and the adition of bentonite to reduce the biodisponibility or the adsorption of LCFA over microbial cell walls, are found to be effective approaches in the operation of fullscale biogas plants. The obtained results reinforce the hypothesis of the adsorptive nature of the LCFA inhibition, and that the recovery process can be followed as an increase in the microbial activity. The nature of the reported microbial activity improvement after subsequent sytem inhibition is characterized by three different techniques: 1) the study of specific microbial activities on different model substrates, 2) the application of molecular biology tools to monitor the microbial population structure and, 3) the development of kinetic expressions of the LCFA inhibition phenomena, based on the adsorption process, within the framework of ADM1 model of the International Water Association. The combined analysis of those confirmed that inhibition and adaptation phenomena are explained by a specific microbial growth, including adsorption in the metabolic LCFA inhibition process. The adsorption-inhibition process is evaluated in detail by determining LCFA adsorption isotherms on granular sludge, LCFA toxicity test, and fluorescence microscopy techniques. This multidisciplinary approach results in the definition of an inhibition preventing strategy based on the introduction of competitive adsorbents, and on stating the importance ofpalmitate during ß-oxidation of LCFA. This study contributes to the understanding of slaughterhouse wastes anaerobic treatment, the LCFA inhibition process, and the biomass adaptation phenomena. The physical adsorption process has been directly related with the LCFA metabolic inhibition, and a new mathematical kinetic expression is proposed. New strategies guiding the operation of anaerobic reactors are suggested in order to obtain high renewable energy yields from slaughterhouse wastes digestion.

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA, 20:4ù6), eicosapentaenoic acid (EPA, 20:5ù3) and docosahexaenoic acid (DHA, 22:6ù3) have been shown to have many health benefits, some of which include lowering blood pressure, providing protection against cardiovascular diseases and improving brain and eye functions. Entomopathogenic fungi, a group of fungal pathogens able to infect insects, were previously reported to produce substantial amounts of VLCPUFAs, however the genes involved in the biosynthesis of these fatty acids have yet to be identified. This research started with fatty acid analysis of five entomopathagenic fungi, of which Conidiobolus obscurus and Conidiobolus thromboides were found to produce high levels of VLCPUFAs such as ARA and EPA. Thus, these two fungal species were selected as potential gene sources for the enzymes involved in the biosynthesis of VLCPUFAs. Using degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) methods; we cloned two full-length putative ∆6 desaturase cDNAs (CoD6 and CtD6) from the two fungi.<p> Functional expression of CoD6 in Saccharomyces cerevisiae showed it codes for a functional Ä6 desaturase, which can introduce a Ä6 double bond into linoleic acid and á-linolenic acid, respectively. However, expression of CtD6 in S. cerevisiae showed it does not have any Ä6 desaturase activity. Using degenerate RT-PCR and RACE, we also cloned two full-length ∆6 elongase cDNAs (CoE6 and CtE6) from the C. obscurus and C. thromboides species. Functional expression of these genes in S. cerevisiae showed CoE6 and CtE6 code for functional ∆6 elongase. Substrate specificity analysis indicated that both preferentially elongate 18-carbon Ä6 desaturated fatty acids, such as ã-linolenic acid and stearidonic acid. In addition, CtE6 can also elongate 20-carbon VLCPUFAs, such as ARA and EPA. The entire eicosatetraenoic acid (ETA, 20:4ù3) biosynthetic pathway was reconstituted in yeast using four genes, CoD6 (a ∆6 desaturase) and CoE6 (a ∆6 elongase) from Conidiobolus obscurus, CpDes12 (a Ä12 desaturase) and CpDesX (a ù3 desaturase) from Claviceps purpurea. Yeast transformant expressing the four genes produced several new fatty acids. Among them, eicosatetraenoic acid (ETA) accounts for approximately 0.1% of the total fatty acids. Although the level of ETA in the transformant is low, this represents the first report describing the reconstitution of the entire ETA pathway in yeast without exogenous supplementation of any fatty acids.

The biosynthesis of wax components containing secondary functional groups was investigated in the current study. Two fundamentally different pathways were proposed to introduce the secondary functional groups. One pathway involves hydroxylation of elongated substrates. Wax components characterized by two functional groups located on or near the centre of the carbon chain, nonacosane-14,15-diol, -14,16-diol and -13,15-diol as well as corresponding ketols were identified for the first time in Arabidopsis stem wax. The alkanediols and ketols were dominated by the C-14,15 isomers. The absence of alkanediols and ketols in Arabidopsis mah1 mutants that are deficient in secondary alcohol biosynthesis confirmed the biosynthetic relationship between secondary alcohols and alkanediols/ketols (Chapter 3). In pea (Pisum sativum) leaf wax, two novel compound classes were identified as primary/secondary alcohols dominated by octacosane-1,14-diol and secondary/secondary alkanediols hentriacontane-9,16-diol, -8,15-diol and -10,17-diol. Co-localization of the secondary/secondary alkanediols and hentriacontan-15-ol and -16-ol pointed to a biosynthetic relationship (Chapter 4). The diverse structures of compounds identified in the current study suggested that hydroxylases can use substrates other than alkanes. The predominance of isomers within homologues indicated a regiospecificity of the hydroxylases involved in wax biosynthesis. In addition to hydroxylation, secondary functional groups could also be introduced through elongation of carbon chains. Homologous series of 5-hydroxyaldehydes (C₂₄ and C₂₆-C₃₆) and 1,5-alkanediols (C₂₈-C₃₈) were identified in yew (Taxus baccata) needle wax. The relative position of both functional groups suggested that these two compound classes are biosynthetically related and their secondary functional groups are introduced during elongation (Chapter 5). The results of incubation of ¹⁴C-labeled malonyl-CoA and acyl-CoAs with different chain lengths in the presence of California poppy (Eschscholzia californica) microsomes provided the first evidence to support the elongation hypothesis. The results indicated that a carbonyl group rather than a hydroxyl group is introduced during elongation. To provide molecular tools for further investigations of the hypothetical pathway, three full length cDNAs encoding putative KCSs were cloned and one of them, PKCSI, was functionally characterized.

Quercetin-3-O-glucoside (Q3G), a glycosylated derivative of quercetin, is a polyphenolic compound known to possess diverse biological activities. Its moderately hydrophilic nature is a critical factor governing the accessibility to the active sites of oxidative damages in vivo. It was hypothesized that biological activities of Q3G can be further enhanced by regioselective acylation with fatty acids which gives more lipophilicity. Q3G was acylated with six selected long chain fatty acids: stearic acid, oleic acid, linoleic acid, ?-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), using Candida antactica lipase. The derivatives were evaluated for their potential in inhibiting lipid oxidation in food systems and human low density lipoprotein (LDL), and cytoprotection and anti-inflammatory effect in cell culture model systems. The fatty acid derivatives of Q3G possessed greater effectiveness in inhibiting lipid oxidation in oil-in-water emulsions, and better cytoprotective effect against H2O2- and cigarette smoke toxicant-induced cytotoxicity when compared to Q3G.

Feeds that maximize reproductive potential are needed to ensure success of captive broodstock. Nutritional requirements for somatic growth of juvenile fishes differ from nutritional requirements of mature fishes and are largely species-specific. Broodstock nutritional research has focused primarily on lipid and fatty acid requirements and their effects on reproductive conditioning due to the importance of these nutrients in providing metabolic energy and structural elements, i.e. for phospholipids for embryonic development. Development of suitable broodstock feeds are limited by not knowing fatty acid requirements for many species. Once requirements are identified, plant, grain, marine, algal, and fungal lipid sources can be blended to develop least-cost diet formulations. The objectives of this dissertation are to (1) evaluate white bass Morone chrysops ovum fatty acid composition and reproductive performance after feeding maternal broodstock graded levels of squid to fish oil; (2) evaluate flax, canola, and corn oils as alternatives to marine oil(s) in white bass maternal broodstock diets; (3) determine extent to which grain oils can replace marine oils in female white bass broodstock diets in order to maintain reproductive performance and quantify fatty acid utilization of larvae with different initial fatty acid profiles; and (4) assess dietary supplementation of 20:4n-6, 20:5n-3, and 22:6n-3 concentrates to boost reproductive performance of female white bass fed primarily plant oil-based lipid sources. Of the marine and plant oils tested, menhaden fish oil provided female white bass broodstock the fatty acids (~3.9% n-3 long-chain polyunsaturated fatty acids; LC-PUFA; dry matter basis) necessary to maximize embryonic survival; however, flax oil, due to its low 18:2n-6 and high 18:3n-3 content, showed promise as a suitable plant oil candidate for partial if not complete marine oil substitution in female white bass broodstock feeds. Differential responses in embryonic and larval survival resulted in comparable total larval yields at 5 days post hatch (DPH) after feeding female broodstock graded levels (0, 33, 67, or 100%) flax to fish oils for 30 weeks prior to spawning. At the end of the endogenous feeding period, fatty acid compositions of flax and fish oil-fed broodstock progeny deviated from initial ova composition. Although n-3 LC-PUFA from menhaden fish oil are essential for embryonic survival, sunshine bass appear to have lower n-3 LC-PUFA requirements after hatch. Larval survival was highly dependent on the presence of C18 PUFA present due to flax oil inclusion in maternal diets. Embryonic survival of progeny produced from broodstock fed dietary saturated fatty acid-rich plant lipids supplemented with intact LC-PUFA concentrates (~3.4% n-3 LC-PUFA; dry matter basis) was similar to that of the broodstock fed the menhaden fish oil control diet containing 4.8% n-3 LC-PUFA. Although the dietary requirement for n-3 LC-PUFA was reduced by feeding these LC-PUFA concentrates in combination with plant lipids, menhaden fish oil is still the most viable option for least cost broodstock diet formulations intended for white bass.

Seafood represents the most important source of long-chain polyunsaturated fatty acids (LC-PUFAs) in the human diet. However, consuming fish can present risks from persistent organic pollutants (POPs) that bioaccumulate in edible tissues following dietary exposure. In farmed fish, POPs accumulate as a result of feeding diets based on fish oil (FO). Fish oil substitution can reduce POP accumulation, but also results in loss of beneficial LC-PUFAs. Fish oil-based finishing diets at the end of production can restore LC-PUFAs, but this strategy also increases POPs. The present study assessed the use of saturated fatty acid (SFA)-rich lipids to replace fish oil in grow-out feeds in conjunction with a fish oil-rich finishing diet to determine if this strategy could produce hybrid striped bass with equal production performance, equivalent LC-PUFA levels, and reduced POP concentrations. Triplicate tanks of hybrid striped bass were raised on diets containing fish oil (100% FO), fish oil spiked with additional POPs (100% FO Spike), or blends (50/50 or 25/75) of FO and coconut (CO) or palm (PO) oils (50% CO, 50% PO, 75% CO, 75% PO) with and without an eight week finishing period with the 100% FO diet prior to harvest. Production performance, fillet LC-PUFA, and POP content were assessed. Production performance was not adversely affected by any of the feeding regimens. However, fatty acid profile was altered, with fillets of fish consuming less fish oil having lower LC-PUFA and POP levels. Finishing yielded a modest increase in fillet LC-PUFAs and POPs, but POPs accumulated more readily than LC-PUFAs during finishing. However, harvest fillet POP and LC-PUFA levels in the experimental groups were lower relative to levels in the 100% FO group. Replacing fish oil in aquafeeds can produce fish with reduced LC-PUFAs, and also reduced POPs. Feeding fish oil results in more rapid accumulation of POPs than LC-PUFA. Overall, the 75% fish oil replacement feeds yielded fish with the highest ratio of LC-PUFAs to POPs. Despite lower LC-PUFA content, fillets of fish fed the 75% fish oil replacement feeds could be incorporated into a weekly meal plan with other dietary sources of LC-PUFAs to meet dietary recommendations for these essential nutrients.

Este trabalho teve o objetivo de avaliar o uso de monensina, levedura e a interação destes aditivos no metabolismo digestivo e desempenho de animais Nelores. Foram realizados dois experimentos. Experimento 1. Foram utilizados quatro novilhos Nelores com cânulas no rúmen e no duodeno para avaliar a degradabilidade, digestibilidade, biohidrogenação, microrganismos ruminais e emissão de metano quando fornecidos monensina e/ou levedura na dieta contendo óleo de girassol em experimento quadrado latino e arranjo fatorial 2x2. Os tratamentos consistiram em com e sem levedura e com e sem monensina. A levedura utilizada foi a Sacharomyces cerevisiae cepa 1026. As variáveis estudadas foram degradabilidade in situ, digestibilidade in vivo avaliada pelo método de indicador interno FDNi, pH, nitrogênio amoniacal e ácidos graxos de cadeia curta do rúmen, protozoários ciliados, biohidrogenação ruminal, e estimativa do número das bactérias Anaerovibrio lipolyica, Butyrivibrio fibrisolvens e Megasphaera elsdenii, por PCR em tempo real. Experimento 2. Foi realizado ensaio para avaliar o desempenho e características de carcaça e carne de touros Nelores utilizando os aditivos supracitados. Foram utilizados cinquenta e dois touros Nelores com aproximadamente 233kg em delineamento em blocos ao acaso. O fornecimento de monensina reduziu o número de protozoários ciliados no rúmen, aumentou a eficiêcia alimentar e a porcentagem de C18:1 trans10-11 e de C18:2 t11c15 na carne. O uso de levedura aumentou a degradabilidade efetiva da MS, o NDT da dieta, os protozoários ciliados, o pH ruminal, a expressão relativa da bactéria B. fibrisolvens, a ingestão de MS dos animais confinados, os ácidos graxos insaturados no conteúdo duodenal e os ácidos graxos saturados na carne, e reduziu a eficiência alimentar, os ácidos graxos saturados no conteúdo duodenal e os ácidos graxos insaturados na carne. Houve interação para a digestibilidade da FDN, ingestão de matéria seca por quilo de peso metabólico, espessura de gordura subcutânea, e ácidos graxos insaturados na carne. O uso de monensina em dieta com óleo foi eficiente para melhora na conversão alimentar. A utilização de cultura de levedura mesmo em dieta com óleo favoreceu os microrganismos ruminais e a energia da dieta. / This work had the objective of evaluate the use of monensin, yeast and the interaction of these additives on Nellore digestive metabolism and performance. It were conducted two trials. Experiment 1. It were utilized four Nellore steers with rumen and duodenum cannulas to evaluate the diet degradability, digestibility, biohydrogenation, rumen microrganisms and methane emission when supplied monensin and/or yeast on the diet with sunflower oil in a latin square desing and a 2x2 fatorial arranjement.The treatments were with and without yeast and with and without monensin. The yeast utilized was the Sacharomyces cerevisiae 1026 strain. The variables studied were in situ degradability, in vivo digestibility evaluated by indicator method NDFi, pH, ammonia nitrogen and rumen short chain fatty acid, ciliate protozoa, biohydrogenation, and estimative of bactéria Anaerovibrio lipolyica, Butyrivibrio fibrisolvens e Megasphaera elsdenii, by real time PCR. Experiment 2. A trial was realized to evaluate the performance and carcass and meat characteristics of Nellore steers utilizing the additives above. It were utilized fifthty two Nellore bulls with 233kg live weight average in a random blocks design. The monensin supplied decreased the protozoa number, the relative efficiency of B. fibrosolvens, increased feed efficiency and the percentage of C18:1 trans10-11 and C18:2 t11c15 in the meat. The use of yeast increased the effective degradability of DM, the TDN, ciliate protozoa, rumen pH, DM intake, unsaturated fatty acid on the duodenal content, and the saturated fatty acid in the meat, and decreased the feed efficiency, saturated fatty acid on the duodenum content and the unsaturated fatty acid of the meat. There were interaction effect to NDF digestibility, dry matter intake per kg of metabolic weight, fat thickness, and unsaturated fatty acid in the meat. The use of monensin was efficient to get the feed efficiency better. The utilization of yeast culture, even in a oil diet, was favorable to rumen microorganisms and the diet energy.

The biosynthesis of wax components containing secondary functional groups was investigated in the current study. Two fundamentally different pathways were proposed to introduce the secondary functional groups. One pathway involves hydroxylation of elongated substrates. Wax components characterized by two functional groups located on or near the centre of the carbon chain, nonacosane-14,15-diol, -14,16-diol and -13,15-diol as well as corresponding ketols were identified for the first time in Arabidopsis stem wax. The alkanediols and ketols were dominated by the C-14,15 isomers. The absence of alkanediols and ketols in Arabidopsis mah1 mutants that are deficient in secondary alcohol biosynthesis confirmed the biosynthetic relationship between secondary alcohols and alkanediols/ketols (Chapter 3). In pea (Pisum sativum) leaf wax, two novel compound classes were identified as primary/secondary alcohols dominated by octacosane-1,14-diol and secondary/secondary alkanediols hentriacontane-9,16-diol, -8,15-diol and -10,17-diol. Co-localization of the secondary/secondary alkanediols and hentriacontan-15-ol and -16-ol pointed to a biosynthetic relationship (Chapter 4). The diverse structures of compounds identified in the current study suggested that hydroxylases can use substrates other than alkanes. The predominance of isomers within homologues indicated a regiospecificity of the hydroxylases involved in wax biosynthesis. In addition to hydroxylation, secondary functional groups could also be introduced through elongation of carbon chains. Homologous series of 5-hydroxyaldehydes (C₂₄ and C₂₆-C₃₆) and 1,5-alkanediols (C₂₈-C₃₈) were identified in yew (Taxus baccata) needle wax. The relative position of both functional groups suggested that these two compound classes are biosynthetically related and their secondary functional groups are introduced during elongation (Chapter 5). The results of incubation of ¹⁴C-labeled malonyl-CoA and acyl-CoAs with different chain lengths in the presence of California poppy (Eschscholzia californica) microsomes provided the first evidence to support the elongation hypothesis. The results indicated that a carbonyl group rather than a hydroxyl group is introduced during elongation. To provide molecular tools for further investigations of the hypothetical pathway, three full length cDNAs encoding putative KCSs were cloned and one of them, PKCSI, was functionally characterized. / Science, Faculty of / Chemistry, Department of / Graduate

Abstract Very long chain fatty acids (VLCFA) are essential molecules that take part in many different cellular processes such as membrane pore stabilization, membrane trafficking and signaling pathways. The fatty acid elongation pathway in yeast has been studied for about a decade. As part of our work on cellular VLCFA elongation, we identified and characterized the condensing enzyme as well as ketoacyl reductases of the elongation pathway in cotton. In order to identify the yeast 3-hydroxyacyl-CoA dehydratase, we introduced a redundancy in this function by engineering a chimera consisting of the two first predicted transmembrane domains of Elo3p and the hydratase2 domain of Candida tropicalis Mfe2p. Yeast harboring the chimeric construct were subjected to random mutagenesis, and screened for mutants whose survival was dependent on the chimera. The mutants isolated contained RFT1 mutations and exhibited a defect in protein glycosylation, but no VLCFA deficiencies. The N-linked glycosylation pathway is well conserved in eukaryotes. Glycan synthesis occurs on the ER membrane; first on the cytoplasmic side up to Dol-PP-GlcNAc2Man5, which is then translocated to the ER luminal side in an Rft1p-dependent flipping process. The core glycan is further extended to Dol-PP-GlcNAc2Glc3Man9, and then transferred to an asparagine side chain of the nascent polypeptide to be glycosylated. It was found that the Elo3'-hydratase2 chimera acts as a multicopy suppressor of the Rft1p deficiency. The subsequent studies elucidated new aspects of Rft1p function, as well as a hitherto under-appreciated role of the ER associated protein degradation process in the maintenance of ER integral membrane complexes and the physical integrity of the membrane. The functionality of the human Rft1p homologue was demonstrated using a yeast complementation assay. A mutant variant from a patient was analyzed, aiding in the identification and characterization of the first reported case of a glycosylation deficiency in humans caused by a defective RFT1 allele.