Apparent ileal amino acid digestibility and the nutritive value of the triticale cultivars Moreno and Ulrika for growing-finishing pigs

Selostus: Ruisvehnälajikkeiden Ulrika ja Moreno rehuarvo lihasikojen ruokinnassa

Composition, ileal amino acid digestibility and nutritive value of organically grown legume seeds and conventional rapeseed cakes for pigs

Selostus: Palkoviljojen ja rypsipuristeiden koostumus, aminohappojen ohutsuolisulavuus sekä rehuarvo sikojen ruokinnassa

The effects of mutated cationic amino acid transporter y+LAT1 at the cellular and systemic level

y+LAT1 is a transmembrane protein that, together with the 4F2hc cell surface antigen, forms a transporter for cationic amino acids in the basolateral plasma membrane of epithelial cells. It is mainly expressed in the kidney and small intestine, and to a lesser extent in other tissues, such as the placenta and immunoactive cells. Mutations in y+LAT1 lead to a defect of the y+LAT1/4F2hc transporter, which impairs intestinal absorbance and renal reabsorbance of lysine, arginine and ornithine, causing lysinuric protein intolerance (LPI), a rare, recessively inherited aminoaciduria with severe multi-organ complications. This thesis examines the consequences of the LPI-causing mutations on two levels, the transporter structure and the Finnish patients’ gene expression profiles. Using fluorescence resonance energy transfer (FRET) confocal microscopy, optimised for this work, the subunit dimerisation was discovered to be a primary phenomenon occurring regardless of mutations in y+LAT1. In flow cytometric and confocal microscopic FRET analyses, the y+LAT1 molecules exhibit a strong tendency for homodimerisation both in the presence and absence of 4F2hc, suggesting a heterotetramer for the transporter’s functional form. Gene expression analysis of the Finnish patients, clinically variable but homogenic for the LPI-causing mutation in SLC7A7, revealed 926 differentially-expressed genes and a disturbance of the amino acid homeostasis affecting several transporters. However, despite the expression changes in individual patients, no overall compensatory effect of y+LAT2, the sister y+L transporter, was detected. The functional annotations of the altered genes included biological processes such as inflammatory response, immune system processes and apoptosis, indicating a strong immunological involvement for LPI.

Prediction of Phosphorylation Sites from Protein Sequences

Phosphorylation is amongst the most crucial and well-studied post-translational modifications. It is involved in multiple cellular processes which makes phosphorylation prediction vital for understanding protein functions. However, wet-lab techniques are labour and time intensive. Thus, computational tools are required for efficiency. This project aims to provide a novel way to predict phosphorylation sites from protein sequences by adding flexibility and Sezerman Grouping amino acid similarity measure to previous methods, as discovering new protein sequences happens at a greater rate than determining protein structures. The predictor – NOPAY - relies on Support Vector Machines (SVMs) for classification. The features include amino acid encoding, amino acid grouping, predicted secondary structure, predicted protein disorder, predicted protein flexibility, solvent accessibility, hydrophobicity and volume. As a result, we have managed to improve phosphorylation prediction accuracy for Homo sapiens by 3% and 6.1% for Mus musculus. Sensitivity at 99% specificity was also increased by 6% for Homo sapiens and for Mus musculus by 5% on independent test sets. In this study, we have managed to increase phosphorylation prediction accuracy for Homo sapiens and Mus musculus. When there is enough data, future versions of the software may also be able to predict other organisms.

Inactive endosialidase-based detection of bacterial and oncofetal polysialic acid

Polysialic acid is a carbohydrate polymer which consist of N-acetylneuraminic acid units joined by alpha2,8-linkages. It is developmentally regulated and has an important role during normal neuronal development. In adults, it participates in complex neurological processes, such as memory, neural plasticity, tumor cell growth and metastasis. Polysialic acid also constitutes the capsule of some meningitis and sepsis-causing bacteria, such as Escherichia coli K1, group B meningococci, Mannheimia haemolytica A2 and Moraxella nonliquefaciens. Polysialic acid is poorly immunogenic; therefore high affinity antibodies against it are difficult to prepare, thus specific and fast detection methods are needed. Endosialidase is an enzyme derived from the E. coli K1 bacteriophage, which specifically recognizes and degrades polysialic acid. In this study, a novel detection method for polysialic acid was developed based on a fusion protein of inactive endosialidase and the green fluorescent protein. It utilizes the ability of the mutant, inactive endosialidase to bind but not cleave polysialic acid. Sequencing of the endosialidase gene revealed that amino acid substitutions near the active site of the enzyme differentiate the active and inactive forms of the enzyme. The fusion protein was applied for the detection of polysialic acid in bacteria and neuroblastoma. The results indicate that the fusion protein is a fast, sensitive and specific reagent for the detection of polysialic acid. The use of an inactive enzyme as a specific molecular tool for the detection of its substrate represents an approach which could potentially find wide applicability in the specific detection of diverse macromolecules.

Phylogenetic Analysis of Avidins and Expression of Novel Avidins from Lactrodectus hesperus and Hoeflea phototrophica

Avidins (Avds) are homotetrameric or homodimeric glycoproteins with typically less than 130 amino acid residues per monomer. They form a highly stable, non-covalent complex with biotin (vitamin H) with Kd = 10-15 M (for chicken Avd). The best-studied Avds are the chicken Avd from Gallus gallus and streptavidin from Streptomyces avidinii, although other Avd studies have also included Avds from various origins, e.g., from frogs, fishes, mushrooms and from many different bacteria. Several engineered Avds have been reported as well, e.g., dual-chain Avds (dcAvds) and single-chain Avds (scAvds), circular permutants with up to four simultaneously modifiable ligand-binding sites. These engineered Avds along with the many native Avds have potential to be used in various nanobiotechnological applications. In this study, we made a structure-based alignment representing all currently available sequences of Avds and studied the evolutionary relationship of Avds using phylogenetic analysis. First, we created an initial multiple sequence alignment of Avds using 42 closely related sequences, guided by the known Avd crystal structures. Next, we searched for non-redundant Avd sequences from various online databases, including National Centre for Biotechnology Information and the Universal Protein Resource; the identified sequences were added to the initial alignment to expand it to a final alignment of 242 Avd sequences. The MEGA software package was used to create distance matrices and a phylogenetic tree. Bootstrap reproducibility of the tree was poor at multiple nodes and may reflect on several possible issues with the data: the sequence length compared is relatively short and, whereas some positions are highly conserved and functional, others can vary without impinging on the structure or the function, so there are few informative sites; it may be that periods of rapid duplication have led to paralogs and that the differences among them are within the error limit of the data; and there may be other yet unknown reasons. Principle component analysis applied to alternative distance data did segregate the major groups, and success is likely due to the multivariate consideration of all the information. Furthermore, based on our extensive alignment and phylogenetic analysis, we expressed two novel Avds, lacavidin from Lactrodectus Hesperus, a western black widow spider, and hoefavidin from Hoeflea phototrophica, an aerobic marine bacterium, the ultimate aim being to determine their X-ray structures. These Avds were selected because of their unique sequences: lacavidin has an N-terminal Avd-like domain but a long C-terminal overhang, whereas hoefavidin was thought to be a dimeric Avd. Both these Avds could be used as novel scaffolds in biotechnological applications.