Thirty-five volunteers were randomized and twenty-nine completed the study. Mean plasma and red blood cell (RBC) total DHA concentrations, which were analyzed among all participants as a measure of adherence, increased significantly in the DHA-enriched treatment compared to control oil-control flour. The plasma and RBC n-6: n-3 ratio was reduced after consumption of HOCODHA-control flour compared to control oil- control flour. The present study failed to see differences in body composition with the HOCODHA-barley flour treatment versus control oil-control flour treatment. In conclusion, significant increases in plasma EPA and DHA levels, as well as the omega-3 index, provide evidence supporting the cardioprotective effects of HOCODHA. The present study demonstrated that in the context of current Western macronutrient intakes, altering the dietary fatty acid composition and adding β-glucan had no major effect on body composition during the 28 days controlled dietary intervention. / February 2017

Tuberculosis emerged from its grave to be one of the deadliest diseases of the present time after recently developing a synergy with AIDS. A fatty acid condensing enzyme-mtFabH has been proposed to connect the key processes involved in biosynthesis of mycolic acids, an important component of mycobacterial cell wall. It condenses long acyl Coenzymes A (CoA; up to C20CoA) with malonyl Acyl Carrier Protein (ACP) to form the elongated β-ketoacyl-ACP which further undergoes rounds of elongation to form mero-mycolate branch of mature mycolic acids. Owing to its proposed central position in mycolic acid synthesis, mtFabH has attracted considerable attention as a good anti-mycobacterial target.In this study, we utilized important biochemical tools such as site directed mutagenesis, mass spectrometry and X-ray crystallography to address some of the key unanswered questions regarding the intricate workings of mtFabH. We solved the first co-crystal structure of substrate C12CoA with mtFabH and further analyzed the substrate specificity of this acylation step. This structure depicts the mode of acyl-CoA binding in mtFabH channels; and its comparison with the parallel E.Coli-acetyl CoA structure provides important similarities and differences in substrate binding in these two FabH enzymes. It also posed an important question about the trajectory of long acyl chain CoA into the deep and "seemingly closed" substrate binding pocket of mtFabH. By utilizing disulfide-based inhibitors, we showed that large conformational changes are necessary to facilitate ligand trafficking in mtFabH while the high catalytic turnover rate of the enzyme is maintained. We also proposed the most likely location of the involved loop.A much faster and less cumbersome assay for mtFabH was also developed and it was utilized to characterize a series of inhibitors. This assay utilizes the commercially available radioactive malonyl-CoA in lieu of malonyl-ACP, the physiological substrate, and thus can serve as ACP independent assay for mtFabH.These studies further our understanding of the biochemistry of mtFabH, which along with the faster assay could be helpful in designing potent mtFabH inhibitors as anti-tubercular agents in the future.

Essential fatty acids nutrition and its effects on immune responses of the juvenile grouper, Epinephelus malabaricus Feng-Cheng Wu (Advisor: Dr. Houng-Yung Chen) Institute of Marine Biology, National Sun Yat-sen University Kaohsiung 804 Taiwan. A series of three experiments was conducted to study the essential fatty acids nutrition and its effects on immune responses (IR) of the juvenile grouper, Epinephelus malabaricus. All experimental diet contained 10 g/100 g diet supplemental lipids from various sources. A reference diet was used in all experiments and contained natural oil mixture of cod liver oil, linseed oil, and safflower oil at a rate of 2:1:1 (wt/wt/wt). In experiment 1, juvenile grouper (11.8 ¡Ó 0.7 g) were fed for 12 wks on one of the seven experimental diets, control diet and the reference diet to investigate dietary requirement for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and effects on IR of grouper. Seven experimental diets contained 1 g/100 g diet of DHA and EPA in various combinations and 9 g/100 g diet of tristearin. The control diet contained 1 g/100 g diet of trilinolenin and trilinolein (3:1, wt/wt). The results indicate that there was a significant difference among dietary treatments in growth, phagocytosis and leucocytes proliferation, when stimulated by Con A and PHA-P but not by LPS. However there was no difference in survival rate and relative liver weight. Enhanced growth was observed when the dietary DHA/EPA was greater than 1, indicating that DHA was superior to EPA in promoting fish growth. DHA is the only member in the family of (n-3) highly unsaturated fatty acid (HUFA) that stimulates phagocytic functions of leucocytes and T cell proliferation of the juvenile grouper. In experiment 2, juvenile grouper (11.3 ¡Ó 0.6 g) were fed for 12 wks on one of the eight experimental diets or the reference diet to investigate dietary requirement for linolenic acid (LNA) and linoleic acid (LA), as well as effects on nonspecific IR of grouper. The test diets were supplemented with LNA or LA at a rate of 1 or 2 g LNA or LA/100 g diet or 2 g/100 g diet of LNA and LA in various ratios (3, 1.4, 0.7 and 0.4). Tristearin was used to fill the lipid supplemental level to 10 g/100 g diet. The results show that enhanced growth and optimal non-specific cellular IR were observed when the grouper were fed on the diet having the highest LNA/LA ratio (3:1) or on the diets supplemented with LNA. But the enhancement was not different (P>0.05) from that of the reference diet group. Thus, incorporating 2 g/100 g LNA/LA (3:1, wt/wt) in diet ensures adequacy of the grouper for essential fatty acid. In experiment 3, juvenile grouper (13.2 ¡Ó 0.9 g) were fed on one of the six experimental diets in the 2 ¡Ñ 3 factorial design or on the reference diet for 12 wks to investigate dietary requirement for (n-3)HUFAs and arachidonic acid (AA), as well as effects on IR of grouper. Two levels of (n-3)HUFAs (1 or 2 g/100 g ) in combination with 3 levels of AA (0, 1 or 2 g/100 g) were tested. The results show an enhanced growth when optimal concentrations of AA and (n-3)HUFA were incorporated to the diets. Liver (n-6)HUFAs concentration reflects IR of the juvenile grouper. Interaction of AA and (n-3)HUFAs in affecting fish growth and IR was insignificant (P>0.05), and concentrations of dietary (n-3)HUFAs or AA did not significantly affect fish survival rate. The results of the 3 experiments show that the grouper will benefit most in growth and IR when their diets contain 1 g/100 g diet of (n-3)HUFA and 1 g/100 g diet of AA, when (n-3)HUFA is a mixture of DHA and EPA at a ratio of 3:1 (wt/wt).

The overall purpose of this thesis is to understand the impact of management on commercial egg layers, whether that be environmental-related, health-related, or other possible stressors within the pullet and post-peak phases. Furthermore, the study seeks to examine what effects to performance and production these impacts may induce within a commercial layer in differing phases, specifically the pullet and post-peak phases. Chapter two explores into coccidiosis within the pullet phase, which induced by a commercial vaccine, can provide stress to a pullet, lowering protection against infection, and seriously compromising its growth and development into peak lay. Chapter three explores into the post-peak phase, a transition from a caged system of production to cage-free system of production was selected. incorporating multiple differing environmental stimuli that can induce stress. If commercial layers prove to possess the capabilities to adapt to these impacts in multiple phases, the possibility of extended production is possible.

Euglena gracilis is a very ancient eukaryote whose chloroplast acquisition and evolution has been independent of higher plants. The organism is unique in possessing two de novo fatty acid synthases, a true multienzyme complex of great size in the cytosol and a plastid-localized type II fatty acid synthase composed of discrete enzymes and acyl carrier protein (ACP). The enzymology of the early steps of fatty acid biosynthesis differed in the Euglena type II fatty acid synthase compared to those of Escherichia coli and plants. The enzymes of Euglena participating in both priming and elongation reactions to form a new carbon-carbon bond were acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and β-ketoacyl-ACP synthase I. The effects of inhibitors on the three different enzymes were noted. All carbon-carbon bond formation was inhibited by cerulenin. Although neither fatty acid biosynthesis nor any of the isolated enzymes were sensitive to diisopropylphosphofluoridate, the three Euglena enzymes studied were sensitive to different sulfhydryl-alkylating agents. Acetyl-ACP supported fatty acid biosynthesis as effectively as did comparable amounts of ACPSH and acetyl-CoA. There was no evidence for a β-ketoacyl-ACP synthase III for priming such as has been reported in type II fatty acid synthase of higher plants and bacteria. The roles of the acetyl-CoA-ACP transacylase and β-ketoacyl-ACP synthase I appear to be unique in the type II fatty acid synthase of Euglena. Acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and β-ketoacyl-ACP synthase I were separated from one another and shown to have different molecular weights.

No / Background Oral administration of the omega-3 fatty acid eicosapentaenoic acid (EPA), as the free fatty acid (FFA), leads to EPA incorporation into, and reduced growth of, experimental colorectal cancer liver metastases (CRCLM). Design: We performed a Phase II double-blind, randomised, placebo-controlled trial of EPA-FFA 2 g daily in patients undergoing liver resection surgery for CRCLM. The patients took EPA-FFA (n=43) or placebo (n=45) prior to surgery. The primary end-point was the CRCLM Ki67 proliferation index (PI). Secondary end-points included safety and tolerability of EPA-FFA, tumour fatty acid content and CD31-positive vascularity. We also analysed overall survival (OS) and disease-free survival (DFS). Results The median (range) duration of EPA-FFA treatment was 30 (12–65) days. Treatment groups were well matched with no significant difference in disease burden at surgery or preoperative chemotherapy. EPA-FFA treatment was well tolerated with no excess of postoperative complications. Tumour tissue from EPA-FFA-treated patients demonstrated a 40% increase in EPA content (p=0.0008), no difference in Ki67 PI, but reduced vascularity in ‘EPA-naïve’ individuals (p=0.075). EPA-FFA also demonstrated antiangiogenic activity in vitro. In the first 18 months after CRCLM resection, EPA-FFA-treated individuals obtained OS benefit compared with placebo, although early CRC recurrence rates were similar. Conclusions EPA-FFA therapy is safe and well tolerated in patients with advanced CRC undergoing liver surgery. EPA-FFA may have antiangiogenic properties. Remarkably, limited preoperative treatment may provide postoperative OS benefit. Phase III clinical evaluation of prolonged EPA-FFA treatment in CRCLM patients is warranted. Trial Identifier: ClinicalTrials.gov NCT01070355. / The Cancer Research UK Clinical Trials Awards and Advisory Committee approved the Trial. PML and ADR were supported by Department of Health/Cancer Research UK Yorkshire Experimental Cancer Medicine Centre funding. The Trial was adopted by the UKCRN Clinical Trials Portfolio (UKCRN ID 8946) allowing West Yorkshire Comprehensive Local Research Network funding of Pharmacy costs. SLA Pharma AG funded some of the experimental work and provided EPA-FFA and placebo. SLA Pharma AG played no role in the design or execution of the Trial. Laboratory costs were also supported by the Leeds Teaching Hospitals Charitable Foundation (Rays of Hope).

The disease malaria afflicts more than a billion people and kills almost one to three million of them every year. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has become worse with the continuous emergence of drug resistance in the parasite. Therefore, improving existing drugs and deciphering new pathways for drug development are the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum (Surolia and Surolia, 2001) has opened up new avenues for the development of new antimalarials as this pathway is entirely different from the human host in which type I pathway exists. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the direct use of these biochemical pathways as targets has emerged so far. This is due to the fact that the structure and function of the targets of these drugs overlaps with that of the human host. A description of such pathways forms the Chapter 1 of the thesis. This is followed by a description of the discovery and the importance of fatty acid biosynthesis pathway and the available literature on the various enzymes that are targets of potential antimalarials. Three isoforms are known for condensing enzymes - FabH which functions in initiation, and FabB and FabF which function in elongation. These isoforms differ in their biochemical properties and have unique roles to play in deciding the membrane composition of any organism. This aspect is also discussed in this chapter. Cloning and expression of -ketoacyl-ACP synthase, FabB/F from Plasmodium falciparum is described in Chapter 2. PfFabB/F is coded by the nuclear genome and is targeted to the apicoplast. The gene is coded by the locus MAL6P1.165 and the putative amino acid sequence of the protein exists in PlasmoDB. All apicoplast targeted proteins have a characteristic bipartite leader sequence consisting of a signal and a transit peptide sequence (Waller et al., 1998). Since the mature protein start site was not known and none of the software packages could predict the site, I aligned the PfFabB/F sequence with the sequences of other -ketoacyl-ACP synthases. On the basis of similarity with E. coli synthases and the mature protein start site of plant synthases, I cloned the first construct of PfFabB/F. The sequence was amplified by PCR and ligated in pET as well as pGEX vector. Expression in various hosts under different temperature and induction conditions could not solubilize the protein in significant quantities and most of the protein was found in inclusion bodies. Next I expressed the sequence with five more amino acids towards the N-terminal and expressed it as an N- terminal NusA fusion. The protein was purified by single step Ni-NTA affinity chromatography. Along with the full length protein (108 kDa), a truncated version of the protein was also obtained. The identity of the protein was confirmed by western blotting using anti-His antibody and anti-FabB/F antibody. In Chapter 3, the substrate specificity of PfFabB/F has been elucidated. PfFabB/F condenses malonyl-ACP with a range of acyl-ACPs. In vivo, acyl carrier protein (ACP) shuttles the acyl substrates between various enzymes of the fatty acid biosynthesis pathway. Enzymes of the pathway other than synthases can accept substrate analogs like acyl-CoA and acyl-NAC’s also in vitro. Acyl-ACPs are not very stable species and thus are not commercially available. Therefore, they have to be synthesized. Since malonyl-ACP could not be synthesized by chemical means, enzymatic synthesis of acyl-ACPs was done. Acyl-ACP synthetase (Aas) or holo-ACP synthase (ACPS) can be used for enzymatic synthesis. Aas is specific only for longer chain substrates; therefore, I decided to use holo-ACP synthase, an enzyme responsible for converting apo-ACP to holo-ACP in the presence of CoA in vivo (Lambalot and Walsch, 1995). When acyl-CoAs are supplied in place of CoA, acyl-ACP is produced. Malonyl-ACP and acyl-ACPs (C4-C16:1) were thus synthesized using holo-ACP synthase from E. coli. The reaction went to almost 95% completion, indicating broad substrate specificity of this enzyme. Bacterial or plant acyl-ACPs of different chain lengths can be resolved by Conformation Sensitive PAGE (Heath and Rock, 1995, Post- Beittenmiller et al., 1991). However, Pfacyl-ACPs synthesized using ACPS did not show any significant shift on CS-PAGE. Therefore I resorted to MALDI-TOF (Matrix Assisted Laser Desorption and Ionization- Time Of Flight) for monitoring the PfFabB/F condensation reactions. PfFabB/F condensed C4-C12-ACPs with malonyl-ACP to their corresponding -ketoacyl-ACP products, with C6, C8 and C10-ACPs being most readily elongated. C14-ACP was very sluggishly elongated, and C16 and C16:1-ACPs were not elongated at all. The condensation reaction was also followed by autoradiography using14C labeled malonyl-ACP, exploiting the clear mobility shift between malonyl-ACP and the other acyl-ACPs. The inhibitory effect of cerulenin, a known inhibitor of condensing enzymes was also checked. PfFabB/F also exhibited malonyl decarboxylase activity resulting in the production of acetyl-ACP in the absence of any significant condensation activity. All the enzymes of fatty acid synthesis pathway required to complete a cycle were assembled together for the in vitro reconstitution of Plasmodium fatty acid synthesis cycle which is described in Chapter 4. Earlier studies of Surolia & Surolia have shown that C12 and C14 fatty acids are the major constituents of Plasmodium lipids. One of my objectives was to determine the maximum length of the acyl ACP product that is synthesized when all the functionally active enzymes of fatty acid synthesis are put together (Kapoor et. al, 2001, Sharma et al., 2003, Karmodiya and Surolia, 2006). Condensing enzymes have a deterministic role in the fatty acid composition as they catalyze the only irreversible step in fatty acid biosynthesis. By analyzing products of the elongation cycle by mass spectrometry it was apparent that C14-ACP is the longest species formed. As already mentioned, PfFabB/F readily elongates C12-ACP but C14-ACP is weakly elongated. Thus the end product of the Plasmodium FAB pathway is influenced by the substrate specificity of PfFabB/F. This confirms the role of PfFabB/F as a decisive enzyme in determining the length of fatty acids synthesized. The inhibition of the cycle by cerulenin and triclosan is also described in this chapter. Chapter 5 describes the ability of the PffabB/F gene to complement for the mutation of condensing enzymes in CY244 cells (fabBtsfabF-, Yasuno et al., 2004). CY244 cells were transformed with pBAD alone or PfFabB/F cloned in pBAD vector (pBADPffabB/F) and the growth was monitored at non-permissive temperature. The product of PfFabB/F could rescue the growth of mutant cells at high temperature but only in the presence of oleic acid. FabB and FabF are the isoforms of condensing enzymes involved in elongation of the fatty acid synthesis cycle but they have a unique role to play (Garwin et al., 1980). FabB is responsible for unsaturated fatty acid synthesis, and fabB-mutants require oleic acid supplementation for growth. FabF is utilized in temperature regulation of membrane fluidity and E. coli FabF elevates the level of C18:1 or cis-vaccenic acid at lower growth temperature but FabF-mutants have no growth phenotype (Ulrich et al., 1983). Rescue of CY244 cells in the presence of oleic acid supplementation indicated that the PffabB/F gene behaves like FabF and not FabB. Analysis of the fatty acid composition of membrane lipids of CY244 cells transformed with pBAD vector or pBADPffabB/F by GC-MS demonstrated no elevated levels of cis-vaccenic acid in transformed cells. This observation is in agreement with the in vitro determined substrate specificity data which shows that PfFabB/F does not elongate C16:1ACP. The thesis ends with a summary of the findings in Chapter 6 in the context of FabB and FabF enzymes known from other sources. 2, 4, 4’-Trichloro-2’hydroxydiphenylether, commonly known as triclosan, has been used as a topical antibacterial agent for decades. I determined its efficacy in treating acute systemic bacterial infection in mouse model. Triclosan, as compared to other well known antibiotics, could extend the survival time of mice by 48 hours. This work is described in Appendix I. (Sharma et al., 2003)

The thesis deals with X-ray crystallographic analysis of two enzymes involved in the fatty acid biosynthesis pathway, known as Fatty Acid Synthase or FAS, of the malarial parasite, Plasmodium falciparum, in order to understand their functions at the atomic level and to provide structural basis for the rational design of antimalarial compounds. Targeting highly specific and well-characterized biochemical pathways to develop effective therapeutic agents has the advantage of designing new drugs or modifying the existing ones based on the details of the known features of the processes. Knowledge of the three-dimensional structures of the molecules involved in the reactions will enhance the capabilities of this procedure. The recently identified fatty acid biosynthesis pathway in Plasmodium falciparum is undoubtedly an attractive target for drug development as it differs from that in humans. In the malarial parasite, each reaction of the pathway is catalyzed by a specific enzyme whereas in humans, the synthesis is carried out by a single multidomain enzyme. Essentially each step in the FAS of P. falciparum can be a potential target to prevent the growth of the parasite as the fatty acids are essential for the formation of the cell membrane which is vital for its survival. All the reactions of this pathway have been well-characterized. Nevertheless, there is a dearth of structural information of the proteins involved in performing various functions in this pathway. When this work was initiated, crystal structures of none of these proteins were reported. The current work on the plasmodial FAS proteins has been undertaken with a view to obtain precise structural details of their reaction and inhibition mechanisms. The introductory chapter of the thesis includes a discussion on malaria, the fatty acid biosynthesis in various organisms and an overview of the structural features of the enzymes involved in the pathway that have been characterized from other organisms.The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the other computational and biophysical methods used to further characterize the enzymes under study. FabI, the enoyl acyl carrier protein reductase, that regulates the third step in FAS has been crystallized as a binary complex with its cofactor NADH and as a ternary complex with NAD+and triclosan. The crystal structures of the binary and the ternary complexes have been determined at 2.5 and 2.2 ˚A, respectively. The mode of binding of the cofactor and the inhibitor triclosan to the enzyme with details of the interactions between them could be clearly obtained from these structures. Each subunit of the tetrameric FabI has the classical Rossmann fold. We carried out a thorough analysis of this structure and compared it with the FabI structures from various sources, four microbial (Escherichia coli, Mycobacterium tuberculosis and Helicobacter pylori) and one plant (Brassica napus), and arrived at a number of significant conclusions: Though the tertiary and the quaternary structures of the enzymes from different sources are similar, the substrate binding loop shows significant changes. The position and nature of the loop are strongly correlated to the affinity of triclosan to the enzyme. Small to major changes in the structure of the enzyme occur to enhance ligand binding. Water molecules play an important role in enzyme-ligand interactions. The crystal structure has also confirmed our previous prediction based on modeling studies of the enzyme that the introduction of bulkier groups at carbon 4’ of triclosan is likely to improve its efficacy as an inhibitor of FabI of P. falciparum. It has also provided the structural basis for its preference to bind to the coenzyme NADH but not to NADPH which was also predicted by our modeling studies. Chapters 3 and 4 discuss the structure solution and a comparative analysis of the crystal structures of FabIs from various sources. The crystal structure of FabZ, the β-hydroxyacyl acyl carrier protein dehydratase of P. falciparum, has been determined at a resolution of 2.4 ˚A. Each subunit of FabZ has a hotdog fold with one long central α-helix surrounded by a six-stranded antiparallel β-sheet. FabZ has been found to exist as a homodimer in the crystals of the present study in contrast to the hexameric form which is a trimer of dimers crystallized in a different condition, reported while we completed the structure of the dimeric form. In the dimeric form, the architecture of the catalytic site has been drastically altered with two catalytic histidine residues moving away from the catalytic site caused by two cis to trans peptide flips compared to the hexameric form. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate-binding. The dimeric state and an altered catalytic site architecture make the dimeric FabZ presented in the thesis distinctly different from the FabZ structures described so far. This is the first observation and report of the existence of an inactive form of the enzyme and its unique structural features. Further analysis using dynamic light scattering and size exclusion chromatographic studies have shown that a pH-related conformational switching occurs between the inactive dimers and active hexamers of FabZ in P. falciparum. These findings open alternate and entirely new strategies to design inhibitors to make FabZ inactive. FabZ crystals show polymorphism with varying length of its longest cell axis. We could collect X-ray diffraction data for three of these forms. An analysis of these forms revealed that three flexible loops of the structure including those containing the peptide flips compete for the space between two symmetry-related molecules. In the form with the longest cell axis, the loops have the highest stability resulting in a better diffraction from the crystal. We also performed docking studies with two previously characterized inhibitors of FabZ. The docking showed that the inhibitors bind strongly at the active site each one making a number of different interactions with the catalytic residues. Observations from our docking studies are in excellent agreement with and strongly supported by chemical modification and fluorimetric analysis of the wild type enzyme and its mutants. Chapters 5 and 6 explain in detail about the structure solution of dimeric form of PfFabZ, the pH induced conformational flipping of two cis-trans peptide flips that lead to different oligomeric states, and the structural basis for these oligomeric shifts. The mechanism of action of PfFabZ inhibitors NAS-21 and NAS-91 are also discussed in detail. Intrigued by the hot dog fold of the Fab enzyme, we have analyzed and compared proteins having this fold in their structures. It has been observed that the fold is often associated with fatty acids. However, the sequences, the quaternary structures, substrate specificities and the reactions that the proteins catalyze are quite diverse. The consensus sequence motifs lining the interface of quaternary association and at active site clearly indicated that the information for different modes of quaternary associations is embedded in the sequences itself. The diversity in function and quaternary association of hot dog fold proteins and their structure-function relationships are discussed in chapter 7. Malaria affects hundreds of millions of people worldwide causing about two million deaths every year. In spite of the commendable success of the available antimalarials, it has not been possible to contain the disease completely as the protozoan has become resistant to a majority of frontline drugs. The structural studies presented here should enhance the current biochemical knowledge to develop selective and more potent inhibitors of the pathway and contribute to the ongoing efforts to fight the disease.

Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, best known for inactivating the signal of N-acylethanolamine (NAE) lipid mediators. In the plant Arabidopsis thaliana, FAAH-mediated hydrolysis of NAEs has been associated with numerous biological processes. Recently, the phylogenetic distribution of FAAH into two major branches (group I and II FAAHs) across angiosperms outside of Arabidopsis (and in other Brassicaceae), suggests a previously unrecognized complexity of this enzyme. Although A. thaliana has long been used to assess biological questions for plants, in this case it will fall short in understanding the significance of multiple FAAHs in other plant systems. Thus, in this study, I examined the role (s) of six FAAH isoforms in upland cotton (Gossypium hirsutum L.) and two FAAHs in the legume Medicago truncatula.

With crude oil demand rising and supplies being depleted, alternative energy, specifically biofuels, are of intense scientific interest. Current plant crop based biofuels suffer from several problems, most importantly the use of land needed for food. Cyanobacteria offer a solution to this problem as they do not compete with land for food and produce hydrocarbons that can be used as biofuels. Upon examination of metabolic pathways competing with hydrocarbon synthesis, it appeared that cyanobacteria lacked the major fatty acid degradative metabolic pathway β-oxidation, generally thought to be a universally occurring pathway. Lack of this pathway in cyanobacteria was confirmed by employing a range of analytical techniques. Bioinformatic analysis suggested that potential enzymes with β-oxidation activity were involved in other metabolic pathways. A sensitive assay was set up to detect acyl- CoAs, the substrates of β-oxidation, using liquid chromatography triple quadrupole mass spectrometry. None could be detected in cyanobacteria. No enzymatic activity from the rate-limiting acyl-CoA dehydrogenase/oxidase could be detected in cyanobacterial extracts. It was found that radiolabeled fatty acids fed to cyanobacteria were utilised for lipid membranes as opposed to being converted to CO2 by respiration or into other compounds by the TCA cycle. An element of the β-oxidation pathway, E. coli acyl-CoA synthetase was ectopically expressed in a strain of cyanobacteria and implications of the introduction of acyl-CoA synthesis were assessed. Finally, the regulation of the fatty acid biosynthetic pathway was investigated. It was determined that under conditions of excess fatty acid, the transcription of acetyl-CoA carboxylase and enoyl-ACP reductase was repressed and acyl-ACP synthetase involved in fatty acid recycling was induced. These results were discussed in relation to fatty acid oxidation and hydrocarbon biosynthesis in other organisms.