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PP1 is a member of the PPP family of serine/threonine phosphatases and has been found in all eukaryotic cells examined to date. The catalytic subunit of PP1 (PP1 c) has been shown to dephosphorylate a wide range of substrates in vitro but its specificity in vivo is determined by a number of regulatory subunits (R-subunits). In Drosophila PP1 c is found to be widely distributed at many discrete sites on third instar polytene chromosomes. These sites primarily overlap with actively transcribing regions, suggesting that PP1 c may regulate chromatin- associated functions, including structure, packaging and transcription. This thesis describes my investigation into the role of nuclear PP1c and a potential regulatory subunit, Protein Phosphatase Type 1 Nuclear Targeting Subunit (PNUTSDm) and the development of a new technique that allows clonal analysis of chromosome spreads from third instar salivary glands. I found that PP1c localises with PNUTSDm isoforms (PNUTSDm-L and PNUTSDm-S) at multiple sites corresponding to actively transcribing chromatin but demonstrate isoform- specific differences. The isoforms were shown to localise to chromatin through different mechanisms with localisation of PNUTSDm-L dependent on RNA or single-stranded DNA- binding. PP1 and PNUTSDm were shown to genetically interact in vivo and mutational \ analysis showed that PP1c and PNUTSDm regulate levels of phospho-histone H3 and phosphorylation of the carboxy-terminal domain of RNAPII during interphase. PNUTSDm- PP1c binding was shown to be necessary for this activity. PP1 c and PNUTSDm mutants were shown to antagonise the function of the histone H3 interphase protein kinase, Jil-1, in a range of developmental processes, and to restore H3Ser10 phosphorylation in JiI-1 mutant oocytes. The results suggest that PP1 may be the first identified interphase histone H3Ser10 phosphatase in Drosophila and that PNUTDm is the regulatory subunit required for this function.

The dietary recommendation to replace saturated fat with carbohydrate as a means of reducing cardiovascular risk, has been hindered by the adverse effect of dietary extrinsic sugars on plasma lipids. The project described in this thesis, formed part of a BBSRC- funded trial to examine the impact of dietary extrinsic sugars on lipoprotein kinetics. A key objective of this project was to design and deliver two diets, high and low in extrinsic sugars, to test the hypothesis of the main trial; that dietary sugars influence plasma lipids, via differential effects on the kinetics of plasma lipoproteins, in groups with moderately raised and low liver fat. This thesis presents the results from five experimental Chapters (2-6): i) An independent, systematic review and meta-analysis of the relationship between dietary sugars and plasma lipids (Chapter 2), that confirmed significant positive and negative associations between dietary sugars, especially fructose, with plasma TAG and HDL-cholesterol, respectively; ii) the development of two experimental diets, high and low in sugars, based on the exchange of starch and sugars, using supermarket foods (Chapter 3). The exchange achieved, and came crose to achieving, target intakes for sugar, that were representative of the upper 95th and lower 5th percentile of sugar intake in the UK; iii) a study of the impact of these two diets on plasma lipids and lipoproteins, in groups with raised and low liver fat (Chapters 4 & 5). The high sugar diet was accompanied by significant increases in plasma TAG, apoproteins (CIII & E), and liver fat, and decreases in HOL-choiesterol, and lipoprotein particle size. In contrast, the low sugar diet was associated with opposite changes in all of these variables. The effects of both diets were shown to be more pronounced in the group with moderately raised liver fat (> 4.2%), and were largely unaltered by the re-analys is of sugar intake by three different methods (Chapter 6). These findings provide evidence for the practical feasibility, and experimental utility of exchanging starch and sugars in real foods. They also provide further support for the recommendation to restrict the intake of dietary extrinsic sugars, especially in individuals with moderately raised liver fat.

The role of glycosylation in the development, regulation, and progression of disease is the focus of considerable research. Glycosylation is a complex posttranslational modification which creates linear or multi-antennary chains of heterogeneous glycans on proteins or lipids. Glycosylation is a tissue specific and highly regulated process modulated by genomic loci encoding specific glycosyltransferases. In light of tissue-specific glycosylation and aberrant glycosylation in a number of disease areas, targeting glycans could potentially lead to selective therapeutic strategies directed at glycoconjugates originating from pathological tissue. However for such a strategy to work, we need to better understand the shape and overall topology of glycan presentation by the glycoprotein which modulates tissue specificity in the extracellular milieu.. Monoclonal antibodies with specificity towards glycan conformations possessing defined biological activity offer promising prospects for this purpose. With this point in view, two naIve .single chain fragment variable (scFv) phage libraries were investigated as potential sources of carbohydrate binding reagents. The defined glycan conformations of the blood group H active mucin-like glycoprotein from human ovarian cyst fluid (HOGS«) were used as target for scFv isolation. A number of different tools were exploited to understand better the nature of HOGS« and its glycans. These include lectins, antibodies, high performance liquid chromatography (HPLG) and mass spectrometry (MS). The purity of HOGS« glycoprotein was assessed by 80S-PAGE and monosaccharide composition determined by HPLG. The sugar linkages and glycan structures were explored by various MS strategies. A solid phase assay approach including inhibition experiments using a panel of 21 plant lectins was used to (1) optimise selection conditions in isolating glycan-specific scFv from the Tomlinson I and J phagemid libraries and (2) to characterise accessible sugar conformations of HOGS«. The Tomlinson I and J libraries were subsequently expanded and screened by selection against HOG544 for glycan specific scFv. SOS-PAGE analysis confirmed the absence of protein impurities and evidence of glycoprotein. HPLG monosaccharide composition analysis identified L-fucose, 0galactose, N-acetyl-:-O-glucosamine and N-acetyl-O-galactosamine. Linkage analysis of partially methylated alditol acetates of depolymerised glycans predominantly showed terminal-fucose, 2-linked galactose and 3-linked galactose amongst other linkages illustrating linkage heterogeneity. Structural analysis and sequencing of permethylated glycans indicates masses correlating to composite .conformations ranging from a trisaccharide to a potential dodecasaccharide and revealed information on branching and core architecture. A substantially modified solid phase assay demonstrated (1) bovine serum albumin is not an optimal blocking agent for detection of glycan on Nunclon Delta surfaces and (2) binding of the lectins Ulex europaeus agglutinin I, Jacalin, Soybean agglutinin, Wheat germ agglutinin and Ricinus communis agglutinin I to HOC544. Inhibition experiments confirmed lectin binding through carbohydrate recognition. An attempt to select for carbohydrate specific reagents using HOC544 as target for the Tomlinson I and J libraries did not produce any functional scFv capable of binding HOC544. The findings of these experiments show substantial heterogeneity in composition, linkage, sequence and overall masses of the oligosaccharides. Despite this complexity in glycan presentation by the glycoprotein, only 5 out of 21 lectins and type-2 H antigen-specific monoclonal antibody were found to bind the glycans of this naturally occurring mucin-like glycoprotein. Na'ive phage libraries may not be the best source for isolating carbohydrate-specific reagent despite their numerous advantages. Understanding the relationship between glycan structure(s) and how it mediates specificity as one entity in recognition events should provide in silico design of carbohydrate binding proteins with a superior approach for developing reagents in comparison to screening complex biological libraries.

The membrane protein P-glycoprotein (Pgp) functions as an active exporter, coupling export of a wide range of drug molecules to ATP hydrolysis. In the absence of full length Pgp crystal structures demonstrating the structural changes which occur during the catalytic cycle, studies which provide insight on the details of structural changes are valuable.

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