It is well established that lipids play an important role in diseases such as non-alcoholic fatty liver disease and cardiovascular diseases. However, in the past decade, it has come to light that lipids may be important in other diseases; particularly in cancer and neurological disorders. Here, lipid metabolism has been investigated using pre-clinical cancer models for melanoma, glioma, non-small-cell lung cancer and colorectal cancer. The role of lipids in the recovery post-stroke has also been studied. Mass spectrometry imaging offers an ideal tool to study lipids in tissue ex-vivo. Lipids ionise well in a number of mass spectrometry modalities, and hundreds of lipids can be imaged in one mass spectrometry imaging experiment. Furthermore, mass spectrometry imaging offers excellent spatial resolution. In this work, both MALDI-MS and DESI-MS have been used for mass spectrometry imaging. Tumour lipid heterogeneity has been a particular focus of this this project. Heterogeneity exists within tumours, as well as between tumours in the same patient; and this causes major problems for therapy. Owing to the untargeted nature, and high spatial resolution of mass spectrometry imaging, it is an excellent technique to study lipid heterogeneity. Adjacent sections (or in some cases the same section used for mass spectrometry imaging), were used for immunofluorescence and H&E staining. By comparing mass spectrometry images with staining techniques, biological reasons for lipid heterogeneity can be established. Here, a particular focus has been on hypoxia (low oxygen tensions), which is a key contributor to tumour heterogeneity, and is associated with aggressive cancers. Additionally, hypoxia is a feature of ischaemic stroke, and lipids in ischaemic stroke have also been investigated. PET is a non-invasive imaging technique which is able to image a radiolabelled molecule (tracer) in the body. Here, PET has been used as a complementary in-vivo technique to mass spectrometry imaging. The tracers [11C] acetate and [18F]-FTHA have been used to image fatty acid synthase and fatty acid uptake in tumours; both of which are hypothesised to be key in cancer progression. REIMS is a newly established mass spectrometry technique. It is ideal for analysing lipids in cells, as sample preparation is minimal. Here, approaches for cell pellet analysis have been tested, and used to detect lipids in cancer cell lines.

A inseminação artificial utlizando sêmen criopreservado de garanhões apresenta uma baixa utilização, principalmente pela variabilidade dos resultados e aumento dos custos relacionados à inseminação. Os lipídeos presentes nas membranas plasmáticas dos espermatozóides participam de diversos eventos associados à fertilização e por sofrerem alterações químicas e estruturais durantes os processos necessários à congelação podem estar relacionados a congelabilidade dos ejaculados. Este trabalho foi composto por dois experimentos. O experimento I utilizou amostras de espermatozóides de seis garanhões e seis touros e objetivou avaliar a possibilidade de análise direta das amostras de espermatozóides e o efeito do armazenamento em metanol por um período de 45 dias a -80°C. As amostras foram coletadas, \"lavadas\" por centrifugações sucessivas e armazenadas nos seguintes meios: PBS Ca++ Mg++ Free, Metanol padrão-HPLC e solução 1:1 PBS Ca++ Mg++ Free e Metanol padrão-HPLC. As amostras em PBS foram analisadas após extração lipídica ou adição de metanol padrão-HPLC previamente à análise. Foi confirmada a possibilidade de análise direta da composição lipídica em MALDITOF, sem necessidade de extração. O armazenamento em metanol ou solução contendo o mesmo não resultou em degradação no período observado, no entanto o armazenamento de amostras previamente extraídas por longos períodos resultaram em resultados alterados. O experimento II utilizou amostras de 16 garanhões, armazenadas em solução 1:1 PBS Ca++ Mg++ Free e Metanol padrão-HPLC. Os ejaculados de origem destas amostras foram submetidos à congelação e os resultados de análise in vitro de motilidade e morfologia espermática foram relacionados com os dados de composição lipídica através da análise dos componentes principais (PCA). Não foi observada nenhuma relação entre os resultados de análise seminal in vitro e a composição lipídica, apesar de alguns autores encontrarem tal relação, principalmente quanto à relação de determinados ácidos graxos e conteúdo total de fosfolipídeos. Estes autores utilizaram técnicas complexas, impossibilitando a aplicação na rotina veterinária a campo. A facilidade de preparo das amostras para MALDI-TOF torna esta técnica viável para análise de composição lipídica, no entanto novos testes devem ser realizados incluindo um maior número de amostas e dados de fertilidade a campo. / Artificial insemination with stallions\' frozen semen have not been widely used, mainly due to the high variability of field results and the increased cost associated with it. Sperm cell membrane lipids play an important role on several events associated with fertilization. These cells go through structural and chemicals changes during freezing and might participate in the freezeability of ejaculates. This work was composed by two experiments. In Experiment I sperm samples from six bulls and six stallions were collected, \"washed\" through centrifugation and stored at -80° in the following mediums: PBS Ca++ Mg++ Free, Methanol HPLC-standard and a solution 1:1 PBS Ca++ Mg++ Free and Methanol HPLC-standard. The samples on PBS were analyzed immediately after lipid extraction or after the addition of methanol. These samples were evaluated onthe day after collection and 45 days later for evaluation of the possibility of a direct analyses with methanol and the effect of long periods of storage with methanol on lipid degradation. The possibility of an extraction-free direct MALDI-TOF analysis was confirmed. The storage with methanol did not resulted in lipid degradation for the period studied, however the storage of previously extract samples resulted on irregular spectrums. In Experiment II 16 sperm samples from stallions were collected and frozen. An aliquot of 500µl from each sample was washed through centrifugation and stored with a 1:1 PBS Ca++ Mg++ Free and Methanol HPLC-standard solution. In vitro sperm analysis of motility and morphology were studied for relations with the lipid profile through a principal components analysis (PCA). It was not observed relation between lipid profile and sperm motility and morphology, as previously reported by others authors. However, these authors methodology were highly complex , making it difficult for field application. MALDI-TOF sample preparation its easy and fast and new test with a larger number of samples and field fertility results may prove this is a viable tool for improving field results with frozen semen artificial insemination.

Abstract not available

Abstract not available

1. The major phospholipids extracted from Bacillus amylolique - faciens were cardiolipin, phosphatidylycerol and phosphatidylethanolamine. 2. The distribution of these phospholipids between the two halves of the cytoplasmic membrane bilayer was studied using phospholipase C ( B. cereus ), phospholipase A2 ( Crotalus ) and the non - penetrating chemical probe trinitrobenzenesulphonic acid ( TNBS ). After treatment of intact protoplasts of B. amylolique - faciens with either phospholipase, approximately 70 % of total membrane phospholipid was hydrolysed ; specifically approximately 90 %, 90 % and 30 % of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin respectively. Under these conditions, protoplasts remained intact and sealed. However, when protoplasts that were permeabilized by cold shock treatment were incubated with either of the phospholipases, up to 80 % of cardiolipin was hydrolysed and phosphatidylglycerol and phosphatidylethanolamine were hydrolysed virtually to completion. In intact cells, 92 % of the phosphatidylethanolamine could be labelled with TNBS under conditions in which the reagent did not penetrate the membrane to any significant extent. 3. These results suggest that 70 % of total phospholipid of this bacillus exists in the outer half of the bilayer. The distribution of phosphatidylethanolamine in this bilayer is highly asymmetric with it being located predominantly in the outer half. The results with phospholipases suggest that the distributions of cardiolipin and phosphatidylglycerol are also asymmetric but independent confirmation or this is required. 4. The fatty acid composition of cells grown at different temperatures was investigated. When cells were grown at 30 ° C, branched - chain saturated fatty acids made up over 80 % of the total fatty acids. Saturated straight - chain fatty acids made up the bulk of the remainder. Less than 1 % of the total fatty acids were unsaturated. Decrease in growth temperature was accompanied by an increase in the ratio of branched to straight - chain fatty acids and a marked increase in the level of unsaturation of branched - chain fatty acids. 5. When cells of this organism, grown at 30 ° C, were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween - 80 to cells caused the critical temperature zone for cold shocking to be significantly lowered. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock. 6. The role of lipids in the process of extracellular enzyme secretion was studied using cerulenin, an antibiotic known to inhibit fatty acid synthesis in microorganisms. Cerulenin inhibited the secretion of alpha - amylase and protease in washed cell suspensions by 80 % and 75 % respectively over 3 hours. The effect was a general one since secretion of all protein species into the medium was drastically reduced by the antibiotic. At the concentration of cerulenin used ( 100 . µ g / ml ), [ 14C ] - acetate incorporation into cellular lipid was inhibited by approximately 50 % but total cellular protein and RNA synthesis were virtually unaffected. The inhibitory effect of cerulenin on alpha - amylase and protease secretion could be partially reversed if cell suspensions were supplemented with either fatty acids prepared from the lipids extracted from B. amyloliquefaciens, or various individual pure fatty acids. These results suggest that fatty acid synthesis may be required for protein secretion by this organism. 7. Attempts were made to detect precursors to extracellular enzymes either associated with the cells or in the culture medium, employing immunological techniques. These experiments, however, were not successful. / Thesis (Ph.D.)--Department of Biochemistry, 1979.

Includes bibliographical references (leaves 169-184) / xiv, 184 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the concept of membrane homeostatis, which implies that biological membranes tend to maintain a constant level of lipid fluidity in the face of potential exogenous and endogenous pertubations. Manipulations of dietary cholesterol and/or saturated (coconut oil) v/s unsaturated (sunflower seed oil) fatty acids have been used to study the relationship between membrane lipid composition, membrane lipid fluidity and membrane-bound enzymes of lipid metabolism; and, to see whether these enzymes act co-ordinately for the maintenance of a membrane homeostatis under these dietary conditions. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1986

Thesis (M.S.)--Pennsylvania State University, 1972. / Includes bibliographical references.

Graduation date: 1994

The biological membrane is composed of lipids and proteins that make up dynamic barriers around cells and organelles. Membrane-spanning proteins are involved in many key processes in the cell such as energy conversion, nerve conduction and signal transduction. These proteins interact closely with lipids as well as with other proteins in the membrane, which modulates and affects their structure and function. In the energy-conversion process, membrane-bound proton-transport proteins maintain an electrochemical proton gradient across the mitochondrial inner membrane or the cytoplasmic membrane of bacteria. This gradient is utilized for ATP synthesis or transport of ions and molecules across the membrane. Results from earlier studies have shown that proton transporters are influenced by their environment. Here, one of these proton transporters, cytochrome c oxidase, has been purified and reconstituted into liposomes or nanodiscs and membrane effects on specific proton-transfer processes were studied. In these studies we observed that the membrane accelerated proton transfer to the surface of cytochrome c oxidase and that there is a protonic link, via a Glu residue that mediates proton transfer from the membrane surface to a proton-transfer pathway in this protein. In addition, the membrane was shown to modulate specific internal electron and proton-transfer reactions. The results from these studies show that the membrane composition influences transmembrane transport. Consequently, our understanding of these processes requires investigation of these transporter proteins in different membrane-mimetic systems of variable and well-defined composition. Furthermore, the data show that membrane surfaces facilitate lateral proton transfer which is presumably essential for maintaining high efficiency in energy conversion. This is particular important in organisms such as alkaliphilic bacteria where the driving force of the electrochemical proton gradient, between the bulk solution on each side of the membrane is not sufficient for ATP synthesis.

There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria Acholeplasma laidlawii and Streptococcus pneumonie and in the plant Arabidopsis thaliana, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures. Here we have determined the genes encoding the main glycolipids of A. laidlawii and S. pneumonie. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of Escherichia coli lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of A. laidlawii and two analogous genes from A. thaliana yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in E.coli membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer in vivo.