Characterization of the Hemagglutinin Cleaving Transmembrane Serine Proteases Matriptase and TMPRSS2

Influenza is one of the commonest infectious diseases affecting millions of people every year including 290,000 – 650,000 heavy casualties. Influenza viruses undergo constant genetic changes and every 10 – 50 years new influenza virus strains emerge that potentially cause a severe pandemic. In this modern interconnected world, experts believe the next influenza pandemic will be a “devastating global health event with far-reaching consequences” [1]. Novel effective anti-influenza drugs are in need. One strategy of influenza research is to focus on host-specific proteases that are essential for virus activation and spread. Trypsin-like serine proteases are crucial for influenza activation by mediating the cleavage of the viral surface glycoprotein HA and hence promoting the fusion potential of the virus. Therefore, their inhibition provides a promising therapeutic approach. The present work focused on the characterization of two relevant HA cleaving type-II transmembrane serine proteases matriptase and TMPRSS2. Chapter 3 and chapter 4 of this thesis engaged with the recombinant production of matriptase (chapter 3) in order to obtain a pure functional enzyme of high quality for a SAR study with novel monobasic (hence potentially bioavailable) matriptase inhibitors of the 3-amidinophenylalanine type (chapter 4). Adequate amounts of high-quality matriptase enzymes were isolated using a new expression system and in total 5 matriptase crystals were available at the end of this thesis for structural analysis. The matriptase inhibitor design in this thesis focused on matriptase-affine compounds with a fair selectivity profile against the blood coagulation enzymes thrombin and fXa. In total, 18 new monobasic and potentially bioavailable, as well as four new dibasic compounds of the 3-amidinophenylalanine types were tested. Based on the last published crystal structure of this inhibitor type in complex with matriptase from 2006 (PDB code 2GV6) docking was used as a structure-based virtual screening method for lead optimization of the compounds N-terminus. Selected compounds were suggested to interact with the carbonyl side chain of Gln175 of matriptase to achieve a higher affinity of matriptase compared to fXa. The 4-tert-butylureido-piperidine could be identified as suitable C-terminus in combination with 3-fluoro-4-hydroxymethyl biphenylsulphonyl N-terminally in order to obtain excellent selectivity over thrombin. The binding mode of this compound (compound 55) was crystallographically determined in complex with matriptase as well as trypsin. Trypsin proved as a suitable alternative to matriptase for detailed binding mode analysis of the compounds N-terminus. However, different preferences were detected for the C-terminus. Dibasic compounds showed higher matriptase affinity and selectivity in comparison with the monobasic analogues. However, the tested monobasic compounds were still decent matriptase inhibitors that are additionally suitable for cell culture and animal studies in their benzamidine prodrug forms, which are well established from related inhibitors of thrombin. In addition, selected monobasic as well as dibasic compounds demonstrated strong suppression of the replication of certain H9N2 influenza viruses in a matriptase-expressing MDCK II cell model. These matriptase inhibitors could be potential lead structures for the development of new drugs against H9 strains for influenza. TMPRSS2 is widely discussed for its role in influenza activation. With a TMPRSS2 dependancy of HA-activation of certain subtypes, the characterization of this protease is an important prerequisite for being available as a target for influenza drug design. However, only little is known about the physiological function of TMPRSS2 and no experimental structure data are available at the moment to enable a structure-based drug development. Therefore, chapter 5 of this thesis focused on the characterization of TMPRSS2 in order to develop a strategy for the isolation of proteolytically active TMPRSS2 from cell culture. Even though, no functional TMPRSS2 could be recovered at the end of this work some new structural characteristics of TMPRSS2 were identified as crucial for functionality insight the cell. In general, TMPRSS2 without the cytosolic part, the transmembrane domain and the LDLRA domain is able to undergo autocatalytically activation if an artificial signal peptide was added N-terminal to enable entry into the endoplasmic reticulum. The presence of the cysteine-rich SRCR domain and the presence of the disulfide chain that connects the SPD and the stem region after activation cleavage have been identified as crucial for activity. N-terminal truncation of TMPRSS2 did not result in obvious dislocation within the cell: as the full-length positive control truncated TMPRSS2 was exclusively found in cell compartments surrounding the nucleus in immunofluorescence experiments. However, a reduced proteolytic cleavage activity towards H3-HA in co-expression experiments has been observed and might be a result of dislocation, since truncated TMPRSS2 is not bound to the biomembrane anymore. In addition, TMPRSS2 has been identified as a potential substrate of matriptase in vitro, which suggests possible participation in several zymogen cascades

Bridging Early Educational Transitions in Learning Through Children's Agency

A longitudinal study of continuity and progression in children's early education reported the significance of the transition from preschool to elementary education for later school success. It was found that the nature of this particular transition is influential for children, parents and educators and therefore for the educational system. An ecological framework was used in order to embrace the complex nature of educational transitions. Educators in 8 preschool and 4 primary school settings, and their managers, were interviewed to explore beliefs about early education. The same educators were observed as the 28 focus children in a cohort of 150 children were tracked during their final year before school and their first year of school, and their written documentation including planning and reports were scrutinised. It was found that despite the similar language used by early educators in early years preschool and primary settings to describe their intentions and motivations for children, there can be major discontinuities between settings, relationships, pedagogy and curriculum and that consequently there are increased challenges for children and for their parents and educators. It is proposed that educators need to collaborate more and to move on from a shared use of terminology to shared meanings, in order to develop a shared conceptual framework which attends to cognitive, social and emotional aspects of children's learning in transition. Certain measurable elements of school progress were noted as part of the local authority's value-added record keeping and it has been possible to make links between educational attainment and other variables. The cohort of children is now entering the last year of primary school education and a number of focus children will now be involved in a study of their transition to secondary education

Female reproductive strategies and mother-calf relationships of common dolphins (Delphinus delphis) in the Hauraki Gulf, New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Zoology at Massey University, Albany, New Zealand

This study reviewed the habitat use, social organisation and behaviour of common dolphin groups categorised by their youngest member, as well as the behaviour of common dolphin calves of different age classes. Most newborn calves were sighted over late spring and early summer, and in water temperatures warmer than for other groups. The water depths at which common dolphin groups were found did not vary according to the age class of their youngest member. However, only groups containing newborns or infants were sighted under 20 metres of water depth. A high percentage of groups encountered contained calves (82.5%). suggesting the importance of the Hauraki Gulf for common dolphins' reproduction. Groups of common dolphins containing calves were found to be of larger size than any other group type. All groups proved to be similarly involved in different behavioural states and showed comparable patterns of association with other species, although groups including newborns were significantly less likely to be seen associated with whales and birds. Groups of common dolphins as a whole did not show a differential reaction to the boat according to the age class of their youngest member. However, mothers and their young calves kept greater distances to the boat than mothers and older calves did. suggesting that the boat may be perceived as a threat during the newborn period. Most of the behaviours that characterise mother-calf relationships varied according to calf age class. The occurrence, frequency, duration and distance of separations increased with older aged calves. A similar increase was found in the time spent without the mother, in the occurrence of association with non-mother dolphins, in the time spent in 'echelon position', and in both mothers' and calves' dive time. Mothers also had longer dive times than calves. Older aged calves tend to present the lowest proportion of synchronous breaths. These changes are likely to represent a gradual increase in calves' independence. Results from this study have extended our knowledge of common dolphins' reproductive ecology, demonstrated that studies of mother-calf relationships in pelagic species of dolphins can he achieved, and allowed future research needs to he identified and management recommendations to be made

Impacts of Internal and External Changing Conditions On French and British Labour Market Segmentation

Drawing on empirical investigations based on national labour force surveys (Enquête Emploi for France, LFS and GHS for The UK) for years 1982-3 and 2001, the aim of this paper is to expose relationships between new external and internal conditions faced by organisations and permanencies and transformations of the macro structure of these two labour markets. We used multiple correspondence analysis (MCA) and hierarchical classification algorithm to constitute the typology of segments structuring labour market. The evidence suggests that the dualistic opposition between a primary and a secondary sector on labour market is still relevant. But in the same time, we go from three segments architecture to a fourfold division on labour market, both in France and the UK, with the apparition of a “Labour Insecurity” class.Labour market segmentation; Dualism; Employment relations

Superconducting order parameter of Sr2_2RuO4_4: a microscopic perspective

The character of the superconducting phase of Sr$_2$RuO$_4$, is topic of a longstanding discussion. The classification of the symmetry allowed order parameters has relied on the tetragonal symmetry of the lattice and on cylindrical Fermi surfaces, usually taken to be featureless, not including the non-trivial symmetry aspects related to their orbital content. Here we show how the careful account of the orbital degree of freedom in Sr$_2$RuO$_4$, leads to a much richer classification of order parameters. We analyse the stability and degeneracy of these new order parameters from the perspective of the concept of superconducting fitness and propose a new best order parameter candidate.Comment: 13 page

How do professionals, parents/carers and children view the role of the learning mentor in a multi-agency setting?

"This project will enable the learning mentor team to establish what links have been made with outside agencies and how professionals view the role of the learning mentor. There is little research locally into this area and it is hoped important learning can be fed back to policymakers and strategists within the local authority." - Page 5

Labour Market Segmentation : a Comparison between France and the UK From the Eighties to nowadays

Regarding changes in French and British labour market as in their educational system since the Eighties, one may address the evolution of their labour market segmentation. Is the predominance of Internal Labour Market in France and Occupational Labour Market in Great Britain (Eyraud, Marsden, Silvestre,1990) still relevant ? We propose a more complex segmentation of labour market with four segments based on tenure, labour mobility and their wage return to account for nowadays situation. Empirical investigations we carried out are based on national labour surveys (Enquête Emploi for France, LFS and GHS for Great Britain). In this paper we expose first investigations and explain which further methods we propose to use in order to characterise French and British labour market segmentation.French labour market; British labour market; labour market segmentation; Internal Labour Market; Occupational Labour Market; United-Kingdom; France