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  • Gerlander, Maija-Liisa (1972)
  • Hallikainen, Pirkko (1951)
  • Ruuska, Helka (1954)
  • Puranen, Anni (Helsingfors universitet, 2011)
    Sodium hydroxide (NaOH) is a strong base which disrupts the seed coat by partial hydrolysis of hemicellulose and lignin in the rumen. NaOH can substitute for mechanical processing of cereal grains. Using total mixed ration including NaOH –treated grains, is an opportunity to avoid the metabolic problems caused by high dietary starch content. The objective of this study was to determine the effects of various levels of NaOH –treated wheat grains in ad libitum total mixed ration diet on feed intake and production of dairy cows. Commonly used oats-barley diet was a control. This study was conducted at the experimental farm of Swedish University of Agricultural Sciences, Umeå, Sweden from September to November 2010. There were 17 multiparous and 6 primiparous cows in the study (Swedish red breed). The cows were kept in a warm loose house barn and intake of total mixed ration was measured by using scale cups. Experimental treatments were ground barley and ground oats in the ratio of 1:1, ground wheat in the ratio of 1:0, ground wheat and NaOH –treated whole wheat in the ratio of 1:1 and NaOH –treated whole wheat in the ratio of 1:0. All the diets were formulated to have a dry matter content of 370 g/kg and crude protein content of 180 g/kg dry matter. Apparent digestibility of nutrient was determined using acid insoluble ash as a marker. Utilization of nitrogen was evaluated using calculated nitrogen balance. The experiment was conducted according to the 4x4 Latin square designs and the statistical differences between the treatments were detected by contrasts. Intakes of dry matter (PQ=0.02) and organic matter (PQ=0.02) increased in pursuance of their improved digestibility as half of the dried wheat was supplemented for NaOH –treated wheat. There was no significant difference between treatments in milk yield or energy corrected milk yield. Milk fat yield (PQ=0.04) and concentration of milk fat increased clearly (PQ=0.004) as half of the dry wheat was substituted for NaOH –treated wheat. By substituting all dry wheat for NaOH -treated wheat, milk protein concentration decreased (PL<0.001). The same occurred for milk urea concentration (PL=0.002). The NaOH –treatment did not result in any economic improvement in this study because concentration of milk protein decreased and intake increased, while milk production remained the same. The best production response was achieved by the mix diet of dried wheat and NaOH –treated wheat.
  • Nurminen, Tuula (2006)
    The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.
  • Jaatinen, Silja (Silja Jaatinen, 2009)
    In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.
  • Hietarinta, Elina (Helsingin yliopisto, 2015)
    Oats is one of the most cultivated grains in the world. Oat contains 5 to 8 % of lipids, which is a lot compared to many other cereals. Most of the oat lipids are triacylglycerols and about 80 % of its fatty acids are nutritionally significant unsaturated fatty acids. Due to high fat content and high amount of unsaturated fatty acids both the processing of oats and the development of new oat products are challenging. Oat lipids and their reactions during the processing and storage are a significant reason for the changes in oat quality and the unpleasant flavour. It is possible to either decrease or increase the stability of lipids with different processing methods. The objective of this study was to examine oat lipid reactions and stability during the storage. Ravintoraisio Oy gave all the samples for the research. There were seven oat products, which all were differently processed. Samples of different ages of these products were analysed. Short-term storage test was made for four samples, over 16 weeks at 40 °C. At first, all the oat samples were milled to small particles and then total lipids of these products were extracted by accelerated-solvent-extraction. Neutral lipid classes, volatile compounds and tocols were measured from the samples. Neutral lipid classes were analysed by the high performance liquid chromatography method with evaporative light scattering detector. Volatile compounds were measured by a solid phase microextraction method with GC-MS. Tocols were measured by the high performance liquid chromatography method with fluorescence detector. All the oat samples contained about 5 % of lipids. Most of the lipids were still triacylglycerols after a long-term storage. Free fatty acids were detected only from non-heat-treated samples. Content of tocols decreased significantly in oat samples during storage. Tocol content decreased when degree of processing rose. The content of oxidation products of oat lipids, like hexanal, also rose during the storage. Hexanal and 2-pentylfuran were the most abundant volatiles in the samples. The highest amount of oxidation products were found in extrudates which were stored for 16 weeks at 40 °C. Based on the results, storing oat products for 16 weeks at 40°C, corresponds with over one year storage at natural storage temperature. The effects of extrusion and heat treatment have strong influence on reactions of oat lipids and storage stability. The lipids of unprocessed oat grains were the most stable. More information is required to identify the exact reason for off-odors and off-flavours.
  • Vainio, Saara (Helsingin yliopisto, 2006)
  • Tigistu-Sahle, Feven (Helsingfors universitet, 2012)
    In addition to being structural components of biological membranes and energy storage of cells, lipids have recently been found to participate as essential players in cell signaling, subcellular transport mechanisms, adjusting functions of integral proteins, and regulation of cell growth and apoptosis. In this study electrospray ionization mass spectrometry (ESI-MS) techniques were used to analyze the phospholipid composition of human bone marrow derived mesenchymal stem cells (BMSC). Numerous chemically distinct lipid species were quantified and the changes in their relative amounts i.e. in the cell’s lipid profile after sequential passaging were followed until senescence (usually from passage 4 up to passage 10, in some cases until p14). Subsequently, the total lipids extracted from the cell pellets were analyzed by triple quadrupole ESI-MS equipment and using lipid-class specific scanning modes. The BMSC lines studied originated from ten donors, five of which were young and five elderly individuals. In culture, the BMSC from both young and aged donors showed time-dependent changes in their phospholipid profiles. The clearest marker findings among individual lipid species were that in phosphatidylcholines (PC) and phosphatidylethanolamines (PE), the species 38:4 (acyl chain pair 18:0/20:4n-6) largely increased towards the late passages, which was seen in the BMSC derived from both the young or aged donors. Thus the reserves of 20:4n-6, the precursor of the eicosanoids having antiproliferative, apoptotic and inflammatory cellular reactions, were increased towards late passages. At phospholipid class level, lysophosphatidylcholine (LysoPC) and phosphatidylinositol (PI) totals, and the ratio of total PI to total phosphatidylserine (PI:PS) were increased from early to latest passages. The results provide new lipid biomarkers to be used for stem cell quality control. The accumulation of polyunsaturated lipid species containing 20:4n-6 or the increase of PI: PS ratio could be potential markers for cell aging and the cells’ poor viability and functionality. The results can be used to develop efficient stem cell therapies and improve patient safety.
  • Tolonen, Nina (Helsingin yliopisto, 2015)
    Background: Cardiovascular disease is the most common cause of death in patients with type 1 diabetes, and the premature mortality rates are especially high in patients with diabetic nephropathy. Diabetic retinopathy is the leading cause of vision loss among the working-age population in industrialized countries. Early identification and aggressive treatment of risk factors are crucial to reduce the incidence of diabetic complications. Aims: To examine the relationships between lipid profiles and diabetic nephropathy, diabetic retinopathy, and incident coronary artery disease (CAD) events in a large nationwide cohort of patients with type 1 diabetes. Subjects and methods: These studies are part of the ongoing Finnish Diabetic Nephropathy Study (FinnDiane). Studies I (N=2927) and III (N=1465) have a cross-sectional design. At follow-up, renal status was verified by a review of all available medical files (Study II, N=2304), and data on CAD events were retrieved from the Finnish Hospital Discharge Register and the Causes of Death Register (Study IV, N=3520). Results: The recommended lipid concentrations of current treatment guidelines were poorly met, especially regarding the target for LDL cholesterol. Triglycerides and apolipoprotein (Apo) B were independent predictors of progression to micro- and macroalbuminuria, and total cholesterol was an independent predictor of progression to end-stage renal disease. HDL and HDL2 cholesterol were independently associated with proliferative diabetic retinopathy (PDR). In patients with PDR, the correlations between albumin excretion rate (AER) and lipid variables were strong. However, in patients without retinopathy no significant correlations were observed. In multivariate models, ApoB, triglycerides, non-HDL cholesterol, ApoB/ApoA-I ratio, and triglyceride/HDL cholesterol ratio were the strongest lipid predictors of an incident CAD event. Conclusions: Lipid abnormalities were associated with an increased risk of all three diabetic complications studied, i.e. diabetic nephropathy, retinopathy, and incident CAD events. Triglycerides and ApoB were independently associated with AER and estimated glomerular filtration rate (eGFR) and predicted the progression to micro- and macroalbuminuria as well as incident CAD events. Far lower concentrations of triglycerides than the currently recommended cut-off level (less than 1.7 mmol/l) increased the risk of progression of renal disease. Total and LDL cholesterol were poor predictors of an incident CAD event in patients with normal AER, in patients with HbA1c below the median (8.3%, 67mmol/l) of the cohort, and in women, in whom the ratios of atherogenic and anti-atherogenic lipoproteins and lipids performed better. Current treatment recommendations may need to be revised to reflect residual CAD risk in patients with type 1 diabetes.
  • Pöhö, Päivi (2013)
    Lipidomics or global lipid profiling is a branch of metabolomics that aims to comprehensive analysis of lipids in a biological system. Lipidomics has become an important research field, since the increased awareness of lipid functions in cell and their role in many common diseases. Also the development of analytical methods, especially mass spectrometry has driven the progress of lipid profiling methods. Lipid profiling allows detection and quantitation of hundreds of intact lipid species in parallel. Challenges in lipidomics are the diversity of different lipid structures and varying lipid concentrations in biological samples. Despite of the development of lipidomics there is not yet a single analytical method to screen all lipids in a biological system. Therefore targeted lipidomics methods are needed in addition to global lipidomics. The literature part of this study presents different analytical methods used in lipidomics studies. The most popular lipid profiling methods are liquid chromatography-mass spectrometry (LC-MS) and direct infusion mass spectrometry or so called shotgun analysis. LC-MS based methods usually utilize reversed phase columns including octadecylsilane stationary phases, but also normal phase chromatography has been applied. In shotgun lipidomics crude extracts are infused directly to mass spectrometer without prior separation. In lipidomics, both unit resolution mass spectrometers like triple quadrupole and ion trap as well as high mass accuracy mass spectrometers like Fourier transform ion cyclotron resonance, Orbitrap, and time of flight mass spectrometers have been used. Mass scanning methods have provided full scan acquisition with accurate mass as well as tandem mass spectrometry like precursor ion scan, neutral loss scan, and multiple reaction monitoring. From the atmospheric pressure ionization techniques, electrospray ionization has been the most applied ionization, but also other soft ionization techniques such as matrix-assisted laser desorption/ionization has been employed. Sample pretreatment in global lipid profiling relies on homogenization and extraction with chloroform-methanol. The most used extraction methods are Folch and Blight and Dyer extractions or modifications of these. In addition, other sample pretreatment methods like solid phase extraction, derivatization, and thin layer chromatography have been used, especially with targeted lipidomics methods. Lipidomics platforms have been applied to several studies of different diseases like diabetes, schizophrenia, and cancer. Many bioactive signaling lipids cannot be detected with lipid profiling methods, since they exist at low concentration and have polar structure. In the experimental part a targeted method was developed for analysis of signaling lipids by ultrahigh-performance liquid chromatography-mass spectrometry. Selected compounds were lysophosphatidic acid, sphingosine-1-phosphate, and phosphoinositides. Method development was mainly done with triple quadrupole mass spectrometer, but also Orbitrap was applied. Several different columns, eluent systems, and pretreatment methods were tested, as well as direct infusion. The best chromatographic separation to signaling lipids was achieved with a reversed phase column at alkaline conditions. However, with this method also several drawbacks were encountered. Peaks were shifting and broadening, there were carryover effects, and problems with repeatability and sensitivity. Direct infusion on the contrary turned out to be problematic because of the unstable electrospray and formation of lysophosphatidic acid from lysophosphocholine in the ionization chamber. Development of a single analytical method for these signaling lipids turned out to be a challenging task and a complete working method for all the studied compounds was not attained.
  • Puustinen, Annika (1993)
  • Kallio, Elisa (Helsingin yliopisto, 2014)
    Periodontitis is characterized by an inflammatory response to bacterial infection in the supporting tissues of the teeth. The disease manifests with gingival swelling and bleeding, increased periodontal pocket depth, and alveolar bone loss. Intact bacteria or bacterial products, including lipopolysaccharide (LPS), may enter the bloodstream through inflamed periodontal tissue or via saliva. Bacterial dissemination, further potentiated by gastrointestinal microbiota, may result in endotoxemia and low-grade inflammation. The general aim of this thesis research was to investigate whether LPS links periodontitis with cardiometabolic disorders. The following topics were studied: genetic factors associated with the susceptibility to periodontitis, the systemic effects of endotoxemia induced by periodontitis and cardiometabolic disorders, as well as the influence of periodontal treatment on plasma LPS activity and lipoprotein composition. A study of genetic polymorphisms of the human major histocompatibility complex region demonstrated that a haplotype comprising six SNPs of the BAT1, NFKBIL1, and LTA genes was associated with the risk of having periodontitis. The risk haplotype showed an association with bleeding on probing, probing pocket depth ≥6 mm, and severe periodontitis, and the result was replicated in two different study populations with concordance. In addition, the serum lymphotoxin-α (LTA) concentration was associated with LTA SNPs of the risk haplotype in homozygous patients, and LTA was expressed in the inflamed periodontal tissue. The systemic effects of the periodontitis-derived endotoxemia were investigated before and after periodontal treatment. In the serum of periodontitis patients, LPS was associated with the proatherogenic very low-density lipoprotein intermediate-density lipoprotein (VLDL-IDL) fraction. Although local healing of the periodontium was successful, the systemic inflammation status of the patients failed to improve after periodontal treatment, reflecting the complexity and persistence of the disease. There were no significant changes in plasma LPS activity or its distribution among lipoprotein classes after periodontal treatment. However, the VLDL of patients with severe periodontitis induced higher expression of proinflammatory cytokines in macrophages when compared with VLDL derived from patients with moderate periodontitis. In addition, VLDL isolated from patients with severe periodontitis with suppuration contained more LPS and induced higher cholesterol uptake in macrophages. The effect of nutrient intake on the association of serum LPS activity with cardiometabolic disorders was examined in a population-based cohort. Endotoxemia was strongly associated with prevalent obesity, metabolic syndrome (MetS), diabetes, and coronary heart disease (CHD). In addition, high serum LPS activity was associated with an increased risk of future CHD events. Even though energy intake was correlated with LPS activity in lean, healthy subjects, the general associations were independent of energy or macronutrient intake. The results indicate that genetic variation in the MHC class III region may be important in periodontitis susceptibility. Endotoxemia and low-grade inflammation originating from periodontitis may induce the proatherogenic properties of VLDL particles via macrophage activation and foam cell formation, thereby promoting atherogenesis. The association of obesity, MetS, diabetes, and CHD with endotoxemia supports the significance of bacterial infections and the immune response in the etiology of cardiometabolic disorders. In conclusion, the findings highlight the close relationship between genetics, the immune response, and lipid metabolism, promoting the role of LPS as a link between periodontitis and cardiometabolic disorders.