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Gallic acid esters (GAE) of varying alkyl chain length were used to determine how antioxidant physical location and partitioning influence hemoglobin-catalyzed lipid oxidation. Specific GAE used were propyl gallate (PG), octyl gallate (OG), and lauryl gallate (LG). GAE partitioning experiments were performed with either isolated cod muscle membranes or washed cod muscle, which primarily contain polar membrane lipids and myofibrillar proteins. Canola oil was used in some experiments to determine how neutral lipids impact partitioning behavior. GAE distribution was determined spectrophotometrically in the recovered membranes, aqueous phase, and oil layer after employing differential centrifugation. Oxidation was monitored by measuring thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides development.When GAE were added to the membrane suspensions, significant differences (p < 0.05) in GAE partitioning were observed in the aqueous phase and membrane sediment, where increases in GAE alkyl chain length corresponded with a decrease in aqueous phase concentrations and increases in the membranes. GAE partitioning in the oil fraction did not show significant differences. Also, increases in GAE alkyl chain correlated with increases in GAE membrane detection when GAE were added to the washed fish muscle (p < 0.05).Adding GAE to the washed cod muscle before the canola oil was the most effective sequence of addition for extending the storage time before lipid oxidation was detected. Among the three GAE tested, PG showed the greatest inhibition against lipid oxidation. The effectiveness of the GAE in the washed cod-canola oil system follows the order, PG > OG > LG, which corresponds with decreasing hydrophobicity.The conclusions of this study are twofold. First, GAE partitioning into the muscle membranes was not the primary factor for delaying the onset of lipid oxidation. Rather, solubility in the aqueous phase showed the greatest impact on extending storage time. Secondly, the order in which GAE and canola oil were added to the washed cod (WC) muscle system influenced hemoglobin-catalyzed lipid oxidation behavior. Adding GAE before the neutral oil may have allowed the GAE to partition more easily into the polar regions of the washed muscle, which in turn provided the most effective protection against oxidation.

In the present study, the ease of rearing, short generation time and molecular research tools available for the zebrafish model (Danio rerio, Hamilton) were exploited to investigate transcriptional regulation in relation to feeding, photoperiod and experimental selection. Chapter 2 describes transcriptional regulation in fast skeletal muscle following fasting and a single satiating meal of bloodworms. Changes in transcript abundance were investigated in relation to the food content in the gut. Using qPCR, the transcription patterns of 16 genes comprising the insulin-like growth factor (IGF) system were characterized, and differential regulation between some of the paralogues was recorded. For example, feeding was associated with upregulation of igf1a and igf2b at 3 and 6h after the single-meal was offered, respectively, whereas igf1b was not detected in skeletal muscle. On the other hand, fasting triggered the upregulation of the igf1 receptors and igfbp1a/b, the only binding proteins whose transcription was responsive to a single-satiating meal. In addition to the investigation of the IGF-axis, an agnostic approach was used to discover other genes involved in transcriptional response to nutritional status, by employing a whole-genome microarray containing 44K probes. This resulted in the discovery of 147 genes in skeletal muscle that were differentially expressed between fasting and satiation. Ubiquitin-ligases involved in proteasome-mediated protein degradation, and antiproliferative and pro-apoptotic genes were among the genes upregulated during fasting, whereas satiation resulted in an upregulation of genes involved in protein synthesis and folding, and a gene highly correlated with growth in mice and fish, the enzyme ornithine decarboxylase 1. Zebrafish exhibit circadian rhythms of breeding, locomotor activity and feeding that are controlled by molecular clock mechanisms in central and peripheral organs. In chapter 3 the transcription of 17 known clock genes was investigated in skeletal muscle in relation to the photoperiod and food content in the gut. The hypothesis that myogenic regulatory factors and components of the IGF-pathway were clock-controlled was also tested. Positive (clock1 and bmal1 paralogues) and negative oscillators (cry1a and per genes) showed a strong circadian pattern in skeletal muscle in anti-phase with each other. MyoD was not clock-controlled in zebrafish in contrast to findings in mice, whereas myf6 showed a circadian pattern of expression in phase with clock and bmal. Similarly, the expression of two IGF binding proteins (igfbp3 and 5b) was circadian and in phase with the positive oscillators clock and bmal. It was also found that some paralogues responded differently to photoperiod. For example, clock1a was 3-fold more responsive than clock1b. Cry1b did not show a circadian pattern of expression. These patterns of expression provide evidence that the molecular clock mechanisms in skeletal muscle are synchronized with the molecular clock in central pacemaker organs such as eyes and the pineal gland. Using the short generation time of zebrafish the effects of selective breeding for body size at age were investigated and are described in chapter 4. Three rounds of artificial selection for small (S-lineage) and large body size (L-lineage) resulted in zebrafish populations whose average standard length were, respectively, 2% lower and 10% higher than an unselected control lineage (U-lineage). Fish from the L-lineage showed an increased egg production and bigger egg size with more yolk, possibly contributing to the larger body size observed in the early larval stage (6dpf) of fish from this lineage. Fish from S- and L-lineage exposed to fasting and refeeding showed very similar feed intake, providing evidence that experimental selection did not cause significant changes in appetite control. Investigation of the expression of the IGF-axis and nutritionally-response in skeletal muscle after fasting and refeeding revealed that the pattern of expression was not different between the selected lineages, but that a differential responsiveness was observed in a limited number of genes, providing evidence that experimental selection might have changed the way fish allocate the energy acquired through feeding. For example, a constitutive higher expression of igf1a was recorded in skeletal muscle of fish from the L-lineage whereas igfbp1a/b transcripts were higher in muscle of fish from the S-lineage. These findings demonstrate the rapid changes in growth and transcriptional response in skeletal muscle of zebrafish after only three rounds of selection. Furthermore, it provides evidences that differences in growth during embryonic and larval stages might be related to higher levels of energy deposited during oogenesis, whereas differences in adult fish were better explained by changes in energy allocation instead of energy acquisition. In chapter 5 the main findings made during this study and their impact on the literature are discussed.