Bacillus subtilis, a model Gram-positive soil bacterium, employs two distinct mechanisms in its membrane adaptation to low temperature: 1) Long-term adaptation to suboptimal temperature is accomplished by increasing the ratio of anteiso- to iso-branched fatty acids in the membrane lipids. 2) After a sudden temperature decrease, the oxygen-dependent fatty acid desaturase (Des) is induced which desaturates fatty-acyl chains incorporated in membrane lipids. The transcription of the gene encoding desaturase, des, is activated by the decrease of the membrane order, via two- component system DesK-DesR. In this work, I studied the influence of cultivation conditions on the mechanisms of B. subtilis membrane adjustments for a low temperature employing fatty acid analysis, fluorescence spectroscopy, differential scanning calorimetry and methods of molecular biology. In the first part of this work, I examined the impact of the cultivation medium on the composition and biophysical features of the B. subtilis cytoplasmic membrane during growth under the optimal (40 řC) and suboptimal (20 řC) cultivation temperature. I compared the nutrient-rich complex medium containing glucose and the mineral medium supplemented with either glucose or glycerol. The results obtained showed the crucial importance of medium...

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The potential for Hibiscus cannabinus L. (kenaf) improvement via genome editing using the CRISPR/Cas9 system to generate gene knock-outs was explored. Studies included target gene identification, target guide RNA (gRNA) selection, plant tissue (explant) choice and media composition for plant regeneration. A putative kenaf phytoene desaturase gene (pds, GEED01047592.1) was identified in the kenaf transcriptome, and molecularly confirmed. Kenaf seedling tissues were transformed via Agrobacterium tumefaciens containing the cas9 gene (endonuclease required for gene knock-out) and each gRNA separately; putative transgenic calli and adventitious shoots arose on a medium containing 1-naphthaleneacetic acid, thidiazuron and silver nitrate. Tissues appeared chlorotic/albino and shoots remained diminutive/dwarf-like. These unique morphologies had also been noted by researchers who successfully knocked out the pds gene in other plant species. Cas9 DNA was detected in these putative transgenic kenaf tissues, but initial DNA sequencing analysis did not confirm knock-out/mutations in targeted areas of the pds gene.

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Microalgae have been proposed as a potential feedstock for biofuel production, and as a result, interest in the biology of these organisms has intensified. These organisms also synthesize lipids that are vital to human health and nutrition. Stress has been shown to have an effect on lipid composition and gene expression in microalgae, but many studies have focused on the effects of abiotic stressors. The purpose of this study was to investigate the effect of biotic stress on lipid-related gene expression in Phaeodactylum tricornutum, a model species of microalgae. The source of biotic stress used in this study was 2E,4E/Z-decadienal, a diatom-derived oxylipin that has been shown to function as a stress signal among diatoms. Real-time RT-qPCR analysis revealed that expression of a patatin-like phospholipase was significantly decreased in decadienal-treated cultures as compared to a solvent control. The expression of a delta-9 desaturase gene believed to be responsible for production of 16:1 fatty acids was increased by a factor of 12. FabI, a gene involved in fatty acid biosynthesis, and PtD5a, which codes for an ER-localized desaturase, were both down-regulated in cells exposed to decadienal. However, changes in expression were only shown to be significant for the patatin-like phospholipase gene. Increased expression of the delta-9 desaturase gene may be a protective mechanism against infection from pathogens, since 16:1 fatty acids have been shown to have antibacterial properties. Regulation of membrane desaturation may also serve to stabilize photosynthetic membranes during times of stress. The down-regulation of the phospholipase gene was surprising, since the release of fatty acids from membrane lipids for oxylipin production is a common response to stress. It is recommended that this experiment be improved upon and expanded in order to determine whether the results obtained are reproducible and how these changes in gene expression correlate with physiological effects. / text

Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability for genes encoding stearoyl-ACP desaturase (SAD), fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6, and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variation. While most of the SNPs and all the indels were silent mutations, both genes carried non-sense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant correlations of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles. / October 2015

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Salicylic acid binding protein 2 (SABP2) that converts methyl salicylate to salicylic acid (SA) plays an obligatory role in the SA-mediated disease resistance pathway in plants. SABP2 interacts with SBIP24 in a yeast two-hybrid screening. SBIP24 belongs to the stearoyl-acyl carrier protein-desaturase protein family. To biochemically characterize the SBIP24, it was cloned from tobacco leaves using RT-PCR and expressed in E. coli. Recombinant SBIP24 was affinity purified using Ni-NTA chromatography. RT-PCR was performed to determine the role of SABP2 in modulating the expression SBIP24. TMV infected transgenic C3 (control tobacco plant containing empty silencing vector) and 1-2 (SABP2-silenced) transgenic tobacco plants were used. Preliminary results indicate that SABP2 may regulate the expression of SBIP24 in tobacco plants. Further studies are needed to confirm these preliminary results.

Recent investigations suggest that reducing stearoyl CoA desaturase (SCD) 1 expression confers protection against obesity and insulin resistance, whereas others show that increasing SCD1 expression protects cells from lipotoxicity. The overall aim of this thesis was to establish the role of SCD1 expression in fatty acid metabolism and insulin stimulated glucose disposal in skeletal muscle. In vitro and in vivo studies were conducted to investigate the relationship between fatty acid subtype, SCD1 expression and fuel metabolism. The role of fatty acid subtype on fatty acid metabolite accumulation and insulin resistance was initially examined in rats. Rats were provided with a low fat diet or a high fat diet consisting of predominantly saturated (SAT) or polyunsaturated fatty acids (PUFA). Rats fed a SAT diet were insulin resistant and had increased skeletal muscle diacylglycerol content whereas rats fed a PUFA diet retained insulin sensitivity and accumulated triacylglycerol rather than diacylglycerol. Interestingly, SCD1 mRNA and protein content were elevated in SAT rats compared with PUFA fed and control fed rats, indicating a possible involvement of SCD1 in the aetiology of insulin resistance. Subsequently, SCD1 expression was examined in the skeletal muscle of various rodent models of genetic and diet-induced obesity. SCD1 content was consistently upregulated in the skeletal muscle of obese rodents. To determine whether SCD1 contributes to or protects from fatty-acid induced insulin resistance, SCD1 levels were transiently altered in L6 skeletal muscle myotubes. Short interfering (si) RNA was used to decrease SCD1 content and a pcDNA3.1/HygromSCD1 vector was introduced to increase SCD1 content. Reducing SCD1 protein resulted in marked esterification of exogenous fatty acids into diacylglycerol and ceramide. Insulin-stimulated Akt (acute transforming retrovirus thymoma) phosphorylation and 2-deoxyglucose uptake were reduced with SCD1 siRNA. Exposure of L6 myotubes to palmitate abolished insulin-stimulated glucose uptake in both control and SCD1 siRNA myotubes. Transient overexpression of SCD1 resulted in triacylglycerol esterification but attenuated ceramide and diacylglycerol accumulation and protected myotubes from fatty acid-induced insulin resistance. Further, these changes were associated with reduced phosphorylation of c-Jun Amino-Terminal Kinase (JNK) and the inhibitor of IêB kinase (IKK), both of which impair insulin signalling. These studies indicated that SCD1 protects from cellular toxicity in L6 myotubes by preventing excessive accumulation of bioactive lipid metabolites. Collectively, these experiments indicate that increasing SCD1 expression may be a protective mechanism designed to prevent insulin resistance in obese phenotypes.

<p>The development of the metabolic syndrome (MetS) and cardiovascular diseases have been suggested to be influenced more by the quality than the amount of dietary fat. The FA composition of serum lipids may be used as biomarkers of dietary fat quality. FAs can, however, also be endogenously synthesized by lipogenic enzymes such as elongases and desaturases. Three desaturases are important in humans: Stearoyl-CoA-desaturase (SCD), ∆6-desaturase (D6D) and ∆5-desaturase (D5D) and surrogate measures of desaturase activities can be estimated as product-to-precursor FA ratios.</p><p>In this thesis, we demonstrated that high SCD, D6D and low D5D estimated activities predicted MetS 20 years later, as well as cardiovascular and total mortality during a maximum of 33.7 years. The relation between D5D and MetS was independent of lifestyle and BMI, while the relation between SCD, D6D and MetS was confounded by BMI. Serum proportions of palmitic (16:0), palmitoleic (16:1) and dihomo-γ-linoleic acids were higher and the serum proportion of linoleic acid (LA) lower at baseline in those individuals who developed MetS. Further, LA was inversely related to mortality, while palmitic, palmitoleic and dihomo-γ-linoleic acids were directly associated with mortality. We also demonstrated that a diet rich in saturated fat “induced” a similar serum FA pattern (including estimated desaturase activities) that was associated with MetS, cardiovascular disease and mortality. We also propose that the SCD ratio [16:1/16:0] might be a novel and useful marker of dietary saturated fat, at least in Western high-fat diets. Finally, genetic variations in the human SCD1 gene were linked to obesity and insulin sensitivity, results that agree with data in SCD1 deficient mice.</p><p>This thesis suggests that dietary fat quality and endogenous desaturation may play a role in the development of metabolic and cardiovascular diseases and the results support current dietary guidelines.</p>