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Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

The study was to investigate the changes in (1) triacylglycerol (TAG) contents and its relationship to (2) lipid synthesis- and metabolism-related enzyme activity and (3) their gene expression in cobia (Rachycentron canadum) during the fast growth period (from October 2006 to April 2007) in ventral muscle and liver in Hsiao-Lu-Chiao island in southwestern Taiwan. The crude lipid was 12% for fed diet, 30-40% for liver while 13% in February and 11% to 9% in other month for muscle. The TAG content of crude lipid was 36 % for fed diets, and from 22% (December) to 40% (February) for muscle, and from 63% (October to February) to 47% (March) for liver. Oil red-O (ORO) staining showed that TAG accumulated in muscle in February but in December in liver. Muscle TAG contents and enzyme activities and mRNA levels of GPDH and FAS increased in February. A decrease in GPDH enzyme activity and mRNA levels but an increase in PEPCK enzyme activity and mRNA levels indicate the increased supply of acetyl-CoA for fatty acid synthesis is in muscle. An increase in FATP2 mRNA levels suggest the influx of fatty acid also contributes to increased fatty acid accumulation in muscle.In liver, TAG and fatty acid contents decreased in March April but increased FAS and PEPCK enzyme activity and mRNA levels. It is possible that fatty acid synthesis is enhanced in March, but a fast transport to other organs results in a net decline in liver fatty acid contents and subsequently a decrease in TAG contents. FATP contents decreased in March-April mRNA, indicating that the influx of fatty acid in decreasing in liver in adult fish. GPDH and GAPDH were not related to lipid metabolism in liver. These data from enzyme activity and mRNA level, demonstrated that a potentially increase in acetyl-CoA via PEPCK contributes to fatty acid synthesis and GPDH-mediated synthesis of G-3-P provide the C skeleton for TAG synthesis.

The term "lipid" covers an extremely diverse range of chemical or molecular species. Lipids, defined as molecules that are sparingly soluble in water but readily soluble in organic solvents, are broadly categorized into "neutral " or "apolar" lipids, and "amphiphilic” or "polar" lipids. Neutral lipids will include simple hydrocarbons, carotenes, triacylglycerols, wax esters, sterol eaters, as wel1 as other lipids such as fatty acids, polyprenols, and sterols In which the hydrophilic function has little Impact on the overall molecular characteristics. Polar lipids include phospholipids, glycolipids, sulfolipids, some sphingolipids, oxygenated carotenoids and chlorophylls.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line.Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally.Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

N-sec -butylthioloated β-lactam (NsβL) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxoacyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that NsβL inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of NsβL on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant NsβL-resistant mutants. Phenotypic analysis of the mutants was performed, as well as sequence analysis of fabH; along with comparative proteomic analysis of intracellular proteomes. Our results indicate that NsβL resistance is mediated by drastic changes in the cell wall, oxidative stress response, virulence regulation, and those pathways associated with CoA. It is our conclusion that Nsβ L has activity towards CoA, resulting in wide-spread effects on metabolism, virulence factor production, stress response, and antimicrobial resistance.

Traumatic brain injury (TBI) has become an increasingly unmet clinical need due to intense military conflicts worldwide. Directly impacted brain cells suffer massive death, with neighboring cells succumbing to progressive neurodegeneration accompanied by inflammatory and other secondary cell death events. Subsequent neurodegenerative events may extend to normal areas beyond the core of injury, thereby exacerbating the central nervous system’s inflammatory response to TBI. Recently CD-36 (cluster of differentiation 36/fatty acid translocase (FAT), a class B scavenger receptor of modified low-density lipoproteins (mLDLs) in macrophages, has been implicated in lipid metabolism, atherosclerosis, oxidative stress, and tissue injury in cerebral ischemia, and in certain neurodegenerative diseases. Accordingly, we proposed that CD-36 has a pivotal role in the neuroinflammatory cascade that further contributes to the pathology of TBI. First, we explored the neuroinflammatory role of CD-36 after acute and chronic stages of TBI. Second, we employed a neuroinflammatory model to test the therapeutic effect of the soluble receptor of advanced end-glycation product (sRAGE); previously shown to abrogate increased CD-36 expression in stroke. Third, we further examined ameliorating TBI related inflammation as a therapeutic pathway by combination of stem cell therapy and sRAGE. At acute stages of TBI, we observed brain co-localization of CD-36, monocyte chemoattractant protein 1 (MCP-1) and ionized calcium-binding adapter molecule 1 (Iba-1) on impacted cortical areas, significant increases of CD-36 and MCP-1 positive cells in the ipsilateral vs. contralateral hemispheres of TBI animals in acute, but no significant increases of Iba-1 expressing cells over time. In early acute stages of TBI immunoblotting showed overexpression of CD-36 in brain cortex when comparing ipsilateral and contralateral hemispheres vs. sham. Spleen CD-36 protein expression at acute post-TBI stages showed no significant difference between TBI and sham groups. In addition, immunohistochemistry revealed minimal CD-36 detection on the cortical area of impact on our chronic group. Spleen immunohistochemistry also showed co-localization of CD-36 and MCP-1 in the red pulp of spleen in acute stages of TBI animals when compared to sham. Ongoing ischemic and hyperlipidemic rodent models suggest that infiltrating monocytes/macrophages from the periphery are the major source of CD-36 in the post-ischemic brain. Likewise, CD-36 expressing monocytes in the spleen after TBI may suggest its role in peripheral immune response, which may exacerbates the inflammatory response after TBI. Therefore, CD-36 may play a key role as a pathological link between inflammation and TBI. Our results suggest an intimate involvement of CD-36 mediated inflammation in TBI, providing novel insights into the understanding of disease neuroinflammation and as a potent therapeutic target for TBI treatment. The critical timing (i.e., 24-48 hours) of CD-36 expression (from downregulation to upregulation) may signal the transition of functional effects of this immune response from pro-survival to cell death. This observed dynamic CD-36 expression also suggests the therapeutic window for TBI. The detection of CD-36 expression in brain areas proximal, as well as distal, to the site of impacted injury suggests its role in both acute and progressive evolution of TBI. CD-36 neuroinflammatory role has clinical relevance for treating patients who have suffered any TBI condition at acute and chronic stages.

Work in the Turos group over the past five years has focused on the development of N-alkylthio beta-lactams, which show antibacterial activity against Staphylococcus (including MRSA), Bacillus, and others. These compounds do not function in the manner of the traditional beta-lactam antibiotics, but were thought to undergo an intracellular thiol-transfer to coenzyme A. In expanding the SAR of these novel compounds, it was found that N-alkylthio-2-oxazolidinones also exhibit antibacterial activity. Although CoA acts as the thiol-redox buffer in the genera most susceptible to the N-alkylthio beta-lactams, studies on Coenzyme A disulfide reductase (CoADR) show that the redox buffer is not affected by these compounds. However, the recent finding that fatty acid synthesis is affected by the N-alkylthio beta-lactams led to the discovery that these compounds act as prodrugs, and that the asymmetric CoA disulfides produced by in vivo thiol transfer are potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III (FabH) through a novel thiol-disulfide exchange with the active site cysteine. Lactams 2a and 2g were also found to be potent inhibitors of this enzyme. In an effort to produce a CoA mixed-disulfide mimic which could cross the cell membrane, a series of simple aryl-alkyl disulfides were synthesized and tested against E. coli, S. aureus, and B. subtilis. Several of these compounds were found to be very potent antibacterials both in vitro and in vivo, with MICs less than 0.125 micrograms/mL. Comparison of the activities of these disulfides with those of acyl-CoA analogs and CoA mixed disulfides support the assertion that FabH is indeed the cellular target of these potent new compounds.

Research and development in aquaculture has supported the knowledge-based development of the sector over the last decades. In particular, species diversification is playing an important role to ensure sustainability of the industry and helping to reduce pressure on wild stocks of those aquatic species for which farming technology is still at the early stages. Due to the increasing pressures on more traditional carnivorous marine finfish species (aquafeed reliance on fishmeal and fish oil, environmental impact, market price) low trophic organisms are receiving more attention to provide sustainable alternatives and integrate production activities with the aim of reducing environmental impacts and to provide secondary high value crops. Integrated Multi-Trophic Aquaculture (IMTA) systems are therefore at the forefront of innovation in the industry. Several invertebrate species have been investigated and tested as integral part of IMTA (mussels, oysters, abalone and macroalgae) and echinoderms have also been considered as good candidates for the future development of this technology. In order to allow for a more widespread uptake of integrated aquaculture, several technical and biological challenges need to be overcome, including a reliable supply of juveniles. In recent years, this has prompted investigation on Echiniculture as a whole and on hatchery technologies in particular. This PhD investigated key constraints in edible sea urchin (Paracentrotus lividus) juvenile production with the aim to improve commercial sea urchin hatchery outputs. The research firstly focused on larval nutrition (Chapter 3 and 4) and specifically tested the hypothesis that larvae required higher dietary inputs of long chain fatty acids than those provided by Dunaliella tertiolecta, a microalgae species widely used in echinoderm larval rearing. Fatty acid composition of P. lividus eggs, investigated in Chapter 3, supported this hypothesis, which was further confirmed by the results obtained in Chapter 4 where microalgae (Cricosphaera elongata, Pleurochrisis carterae and Tetraselmis suecica) with a more balanced fatty acid profile, in particular richer in long chain fatty acids, were employed. This resulted in a significantly improved larval development and survival. Results also indicated that these alternative microalgae species could be successfully grown without modification of the microalgae production protocols in the hatchery where the experimentation had taken place. The third experimental chapter compared static and flow through systems which provides more stable water quality through constant water exchange and reduces larval handling and associated stress. Results indicated that larval survival was significantly improved by the flow-through system and the need for tank cleaning was reduced (three versus seven times per larval cycle when using flow-through and static rearing systems respectively). However, water quality, based on the parameters assessed (NH4, PO4-3, NO2 and NO3), did not show any significant differences between systems. Reduced handling could have therefore played the most important role in promoting larval survival. Both these trials resulted in a significant 5 to 20 % increased survival. A follow-up study, combining flow-through with more suitable microalgae, should be carried out and could result in even further enhanced survival. Then, chapters 6 and 7 focused on broodstock nutrition and subsequent improvement of gamete quantity and quality. These two trials aimed to explore and describe the biological effects that some important nutrients, such as proteins, lipids, fatty acids and carotenoids, have on urchins’ somatic and gonadal growth, gonad biochemical composition during gametogenesis, fecundity and maternal provisioning to developing embryos. Results from the experiment described in Chapter 6 indicated that higher protein content can improve somatic growth in P. lividus adults and that more expensive, protein-, lipid- and energy-rich diets do not significantly enhance fecundity or offspring performance. Results, moreover, highlighted the need for a specifically formulated broodstock diet and gave some insights into what its composition should be, especially in relation to carotenoids. In Chapter 7, fatty acid profiles of P. lividus gonads throughout gametogenesis were studied for the first time. It was observed that, among Long Chain Polyunsaturated Fatty Acids (LC-PUFAs), Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) are primarily accumulated during gametogenesis, whilst Arachidonic acid (ARA) appears to be independent of dietary input. In addition, it was clearly shown that ARA is the only LC-PUFA accumulated in the eggs along with Non Methylene Interrupted Fatty Acids (NMI FAs). As well as looking at the biological effects of different diets on fatty acid profiles of gonadal and larval tissues, the work also expanded on a more fundamental level to explore the metabolic pathway through which precursors could be used by sea urchins for the endogenous production of long chain fatty acids (Chapter 8). Three Expressed Sequence Tags (ESTs) for putative fatty acyl desaturases, one of which was closely related to Octopus vulgaris ∆5-like fatty acyl desaturase, were identified. The newly cloned putative desaturase of P. lividus possessed all typical features of other fatty acyl desaturases. However, because of time constraints, functional characterisation, originally planned, of the new protein could not be performed and further research effort is needed to investigate this important aspect of sea urchin physiology. Overall, the aim of this research project has been achieved as it provided a set of exploitable results and protocols to improve hatchery practices for the production of P. lividus juvenile. However, more research is required to investigate some of the underlying mechanisms behind the observed biological effects such as delay in larval development when T. suecica was used as larval feed, increased broodstock fecundity, improved larval survival in the flow-through system and higher gonadal concentration of some fatty acids (mainly DHA) than provided in the feed.

Natural variation in the relative proportions of saturated and unsaturated fatty acids in seed oils of plants is enormous when considered across a broad taxonomic range of oil seeds. It has been shown that this variation follows a latitudinal cline where the proportions of unsaturated fatty acids increases with increasing latitude as the unsaturated fatty acids in seeds provide energy at a faster rate to germinating seeds at higher latitudes. This variation which follows a latitudinal cline suggests that there may be an adaptive role for this variation. We tested this hypothesis in Arabidopsis thaliana which followed the same trend seen in Helianthus and other angiosperms. In order to understand the underlying genetics of the regulation of the relative proportions of fatty acids and their role in plant evolution, we mapped quantitative trait loci (QTLs) and candidate genes. Here we identified 67 major QTLs responsible for fatty acid synthesis in A. thaliana in Ler-0 x Sha, Ler-0 x Col-4, Ler-2 x Cvi and Ler-0 x No-0 RIL populations. Eight candidate genes were identified based on what is known about seed oil biosynthesis in A. thaliana. Six of the candidate genes collocated to most of the major QTLs. In order to demonstrate that a particular allelic variant is indeed causally related to the phenotype, we investigated DNA polymorphisms in the parental and the RIL line alleles of the collocating candidate genes. Single nucleotide polymorphisms (SNPs) were identified in the collocating candidate genes to study the correlation between the sequence variants and the particular phenotype. We identified 232 SNPs with 77 in the putative regulatory regions upstream of the 5’UTR, 61 in the introns, 18 in the 5’UTR regions, 2 in the 3’UTR regions, and 45 occurring in the exons with 10 non-synonymous substitutions affecting the amino acid residues. We also detected 44 insertions/deletions in the coding, non-coding, 5’UTR, 3’UTR and the regulatory regions. Sequence variation in the fatty acid genes due to SNPs and insertions/deletions should be valuable in tests of association to investigate how the relative proportions of saturated and unsaturated fatty acids are regulated in wild plants and what role they have played in plant evolution and also in breeding oil seed crops that are healthier or have two types of fatty acids in proportions appropriate for different uses. / text