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Synthesis and protein curing abilities of membrane glycolipidsWikström, Malin January 2006 (has links)
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.
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Expression and structure-function characterisation of herpesviral proteinsDahlroth, Sue-Li January 2008 (has links)
In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult. The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role.
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Molecular studies of intra-oocyte phosphatidylinositol 3 kinase (PI3K) signaling pathway in controlling female fertilityDubbaka Venu, Pradeep Reddy January 2009 (has links)
The primordial follicle pool is the main source of developing follicles in the ovary. The length of reproductive life and the onset of menopause are governed by the amount of primordial follicles in the ovary. The genetic factors and molecular mechanisms that maintain the primordial follicles in a dormant and surviving state for the whole of reproductive life are not well understood. The phosphatidylinositol 3 kinase (PI3K) signaling pathways in the oocyte that control oocyte growth and early follicular development are largely unknown. The major aim of this thesis was to investigate the functional role of the intra-oocyte PI3K pathway in the regulation of primordial follicle activation and survival. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a major negative regulator of PI3K. The conditional deletion of Pten in the oocytes of primordial follicles led to the overgrowth of oocytes and activation of the entire pool of primordial follicles. There were higher numbers of activated primordial follicles at postnatal day 8 (PD8) in ovaries lacking PTEN in oocytes; by PD35 all the primordial follicles were activated and all the follicles were depleted by 12 weeks, causing premature ovarian failure (POF). In addition, the rate of follicular death that occurs during sexual maturity is reduced in ovaries that lack PTEN in oocytes. Further mechanistic studies revealed that loss of Pten in oocytes resulted in elevated Akt signaling and upregulation of both expression and activation of ribosomal protein S6 (rpS6). The overactivation of primordial follicles in ovaries that lack PTEN in oocytes is believed to be due to elevated expression and activation of rpS6. PTEN in oocytes is indispensable for the maintenance of primordial follicles in dormancy. To study the role of the intra-oocyte PI3K signaling pathway in controlling the survival and maintenance of primordial follicles, 3-phosphoinositide-dependent protein kinase-1 (PDK1) was deleted in oocytes of primordial follicle. The loss of Pdk1 in oocytes led to the depletion of most primordial follicles around the onset of sexual maturity, causing POF during early adulthood. Furthermore, the activation of Akt, p70 S6 kinase 1 (S6K1), and rpS6 was impaired in oocytes that lacked PDK1. The suppressed PDK1–Akt–S6K1–rpS6 signaling in oocytes appears to be responsible for the loss of primordial follicles. The excessive activation of primordial follicles seen in the absence of Pten in oocytes could be reversed by concurrent deletion of Pdk1. In addition, the elevated activation of Akt and S6K1 in the absence of PTEN in oocytes was not observed in PTEN and PDK1 double mutant mice. Similarly, the hyperphosphorylation of rpS6 in oocytes that lack PTEN was prevented in double mutant mice, which was most likely due to downregulation of S6K1 activation. Thus, inactivation of rpS6 in double mutant mice might be the reason for the prevention of excessive primordial follicular activation and survival. PTEN and PDK1 in oocytes are essential for the maintenance of quiescence and survival of primordial follicles. The molecular network involving PI3K/PTEN–PDK1 signaling in oocyte controls the survival, loss, and activation of primordial follicles, which together govern reproductive aging and determine the length of reproductive life in females. The results of the above studies indicate that the mammalian oocyte serves as the seat of programming of follicular activation and survival.
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Design of Glutathione Transferase Variants for Novel Activities with Alternative SubstratesShokeer, Abeer January 2010 (has links)
Glutathione transferases (GSTs) play a pivotal role in cellular defense, since they are main contributors to the inactivation of genotoxic compounds of exogenous and endogenous origins. Directed evolution was used to improve the catalytic activities of Theta class GST T1-1 toward different substrates. The library was constructed by recombination of cDNA coding for human GST T1-1 and rodent Theta class GSTs, resulting in the F2-F5 generations. The clones were heterologously expressed in Escherichia coli and screened for variants with enhanced alkyltransferase activity. A mutant, F2:1215, with a 70-fold increased catalytic efficiency with 4-nitrophenethyl bromide (NPB) compared to human GST T1-1, was isolated from the second generation. NPB was used as a surrogate substrate of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in order to facilitate the screening process. The catalytic efficiency of the F2:1215 with BCNU had improved 170-fold compared to wild-type human GST T1-1, suggesting that NPB is a suitable model substrate for the anticancer drug BCNU. The sequence of the F2:1215 mutant differs from wild-type human GST T1-1 by three residues; one of these differences is Arg234, which corresponds to Trp in the human enzyme. Upon replacing the Trp234 in the human GST T1-1 with Arg, the resulting mutant (hTrp234Arg) showed enhanced alkyltransferase activity with a wide range of substrates (e.g. haloalkanes and other typical GSTs substrates). The three-dimensional structures of both wild-type human GST T1-1 and hTrp234Arg mutant help to explain the higher activity showed by of hTrp234Arg mutant compared to wild-type enzyme. The reciprocal mutation of the residue 234 in mouse GST T1-1 to that found in human, mArg234Trp, caused a dramatic decrease in the activity of the mouse enzyme to be similar to human GST T1-1. This indicates that residue 234 can be considered as a master switch of activities between human and rodent GST T1-1. Another important residue in the C-terminal helix of GST T1-1 is Met232. Although residue 232 points away from the H-site, it influences the catalytic activity and substrate selectivity of the mouse GST T1-1. A minor modification of Met232 induces major changes in the substrate-activity profile of the mouse GST T1-1 to favor novel substrates such as isothiocyanates and hydroperoxides and decreases the activity toward substrates that catalyzed by the wild-type enzyme.
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The Ins and Outs of Membrane Proteins : Topology Studies of Bacterial Membrane ProteinsRapp, Mikaela January 2006 (has links)
α-helical membrane proteins comprise about a quarter of all proteins in a cell and carry out a wide variety of essential cellular functions. This thesis is focused on topology analyses of bacterial membrane proteins. The topology describes the two-dimensional structural arrangement of a protein relative to the membrane. By combining large-scale experimental and bioinformatics techniques we have produced experimentally constrained topology models for the major part of the Escherichia coli membrane proteome. This represents a substantial increase in available topology information for bacterial membrane proteins. Many membrane protein structures show signs of internal duplication and approximate two-fold in-plane symmetry. We propose a step-wise pathway to explain how proteins with such internal inverted repeats have evolved. The pathway is based on the ‘positive-inside’ rule and starts with a protein that can adopt two topologies in the membrane, i.e. a “dual” topology protein. The gene encoding the dual topology protein is duplicated and eventually, through re-distribution of positively charge residues, the two resulting homologous proteins become fixed in opposite orientations in the membrane. Finally, the two proteins may fuse into one single polypeptide with an internal inverted repeat structure. Finally, we re-create the proposed step-wise evolutionary pathway in the laboratory by showing that only a small number of mutations are required in order to transform the homo-dimeric, dual topology protein EmrE into a hetero-dimeric complex composed of two oppositely oriented proteins.
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CHARACTERISATION OF HEPARAN SULPHATE (HS) FROM MOLE RAT LIVERKelly, Caitríona January 2005 (has links)
This thesis is focused on the heparan sulphate (HS) structure from blind mole rat liver. HS is a glycosaminoglycan that is produced as a proteoglycan, in which linear polysaccharide chains are attached covalently to a protein core. Proteoglycans are widespread molecules in the body and have many important physiological functions. HS is synthesized as a polymer of alternating glucuronic acid and N-acetylglucosamine units. Parts of the polymer are subsequently modified by N-deacetylation /N-sulphation of the glucosamine units, C-5 epimerization of glucuronic acid to iduronic acid and O-sulphation at various positions. The mole rats are from Israel and are of the Spalax ehrenbergi superspecies. Spalax Judaei (S60) has 60 chromosomes and Spalax Galili (S52) has 52 chromosomes. They are both completely blind and spend their entire life underground in hypoxic conditions. Spalax Galili (S52) inhabits the cool-humid Upper Galilee Mountains and Spalax Judaei (S60) inhabits the warm-dry southern regions. There is no current information about the heparan sulphate structure of these animals. The two blind mole rats (S52 and S60) were metabolically labelled with [3H] Glucosamine. The animals were sacrificed and the organs were taken and frozen. The liver was chosen for the purpose of my project. The HS structure was studied using various chromatographic methods such as ion-exchange and gel filtration. Structural analysis of HS indicated that the size of HS from the liver was the same in both species. However, the domain structure differed between the two animals, particularly with regard to sample S52(1) which had obvious differences. This leads to the study of the heparanase cleavage sites. Disaccharide composition analysis identified varying proportions of disaccharide species in S52 and also the possibility of an unknown disaccharide species.
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Hälsoundervisning : Elevers syn på hälsa inom ämnet Idrott och hälsaPersson, Johanna January 2009 (has links)
Fler unga människor än någonsin är idag överviktiga och stress och stressrelaterade symptom drabbar idag allt fler unga. Därför är det viktigt att unga människor får kunskaper om hur de på bästa sätt kan ta hand om sig själva. Syftet med detta arbete är att undersöka hur elever som läser gymnasiets kurs Idrott och hälsa A ser på den hälsoundervisning de får. Detta är relevant för alla som arbetar som idrott och hälsa lärare för att kunna hitta en jämkning mellan kursplan och elevernas tankar och förkunskaper. Genom intervjuer och fokusintervjuer kom jag fram till att eleverna vill lära sig mer om stress och hur man hanterar stress samt om kost. Eleverna tycker däremot att idrottsläraren inte är rätt person att lära ut kunskaper om tobak.
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On the effects of structure and function on protein evolutionIllergård, Kristoffer January 2010 (has links)
Many proteins can be described as working machines that make sure that everything functions in the cell. Their specific molecular functions are largely dependent on their three-dimensional structures, which in turn are mainly predetermined by their linear sequences of amino acid residues. Therefore, there is a relation between the sequence, structure and function of a protein, in which knowledge about the structure is crucial for understanding the functions. The structure is generally difficult to determine experimentally, but should in principle be possible to predict from the sequence by computational methods. The instructions of how to build the linear proteins sequences are copied during cell division and are passed on to successive generations. Although the copying process is a very efficient and accurate system, it does not function correctly on every occasion. Sometimes errors, or mutations can result from the process. These mutations gradually accumulate over time, so that the sequences and thereby also the structures and functions of proteins evolve overtime. This thesis is based on four papers concerning the relationship between function, structure and sequence and how it changes during the evolution of proteins. Paper I shows that the structural change is linearly related to sequence change and that structures are 3 to 10 times more conserved than sequences. In Paper II and Paper III we investigated non-helical structures and polar residues, respectively, positioned in the nonpolar membrane core environment of α-helical membrane proteins. Both types were found to be evolutionary conserved and functionally important. Paper IV includes the development of a method to predict the residues in α-helical membrane proteins that after folding become exposed to the solvent environment. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.
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Development of competence in biochemical experimental work : assessment of complex learning at university level /Bergendahl, Christina, January 2004 (has links)
Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.
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Hälsoundervisning : Elevers syn på hälsa inom ämnet Idrott och hälsaPersson, Johanna January 2009 (has links)
<p>Fler unga människor än någonsin är idag överviktiga och stress och stressrelaterade symptom drabbar idag allt fler unga. Därför är det viktigt att unga människor får kunskaper om hur de på bästa sätt kan ta hand om sig själva.</p><p> Syftet med detta arbete är att undersöka hur elever som läser gymnasiets kurs Idrott och hälsa A ser på den hälsoundervisning de får.</p><p>Detta är relevant för alla som arbetar som idrott och hälsa lärare för att kunna hitta en jämkning mellan kursplan och elevernas tankar och förkunskaper.</p><p> Genom intervjuer och fokusintervjuer kom jag fram till att eleverna vill lära sig mer om stress och hur man hanterar stress samt om kost. Eleverna tycker däremot att idrottsläraren inte är rätt person att lära ut kunskaper om tobak.</p>
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