Skin damage to two new mango cultivars during irradiation and cold storage.

Mangoes can express several skin disorders following important postharvest treatments. Responses are often cultivar specific. This paper reports the responses of two new Australian mango cultivars to some of these treatments. 'Honey Gold' mango develops "under skin browning" early during cold storage. This is thought to be partly caused by a discolouration of the latex vessels which then spreads to the surrounding cells. The symptoms appear to be worse in fruit from hotter production areas and that have been cooled to temperatures below 18C soon after harvest. Current commercial recommendations are to cool fruit to 18C, which limits postharvest handling options. Recent trials have confirmed that delayed or slowed cooling after harvest can reduce under skin browning. The defect may also be associated with physical injury to the skin during harvesting and packing. Irradiation is potentially an important disinfestation treatment for fruit fly in Australian mangoes. The 'B74' mango cultivar develops significant skin damage following irradiation, mainly due to discolouration of the cells surrounding the lenticels. Recent results confirmed that fruit harvested directly from the tree into trays without exposure to water or postharvest chemicals are not damaged by irradiation, while commercially harvested and packed fruit are damaged. Several major harvest and postharvest steps appear to increase lenticel sensitivity to irradiation. Further work is required to develop commercially acceptable protocols to facilitate 'Honey Gold' and 'B74' mango distribution and marketing.

Status assessment of water use research in turf growth and maintenance.

The strategic objectives of Turf Australia (formerly the Turf Producers Association (TPA)) relating to water use in turf are to: • Source and collate information to support the case for adequate access to water for the Turf production and maintenance sectors and • Compile information generated into a convincing communication package that can be readily used by the industry in its advocacy programs (to government, regulators, media etc) More specifically, the turfgrass industry needs unbiased scientific evidence of the value of healthy grass in our environment. It needs to promote the use of adequate water even during drought periods to maintain quality turfgrass, which provides many benefits to the broader community including cooling the environment, saving energy and encouraging healthy lifestyles. The many environmental, social and health benefits of living turfgrass have been the subject of numerous investigations beyond the scope of this review. However further research is needed to fully understand the economic returns achievable by the judicious use of water for the maintenance of healthy turfgrass. Consumer education, backed by scientific evidence will highlight the “false economy” in allowing turfgrass to wither and die during conditions which require high level water restrictions. This report presents a review of the literature pertaining to research in the field of turf water use. The purpose of the review was to better understand the scope and nature of existing research results on turf water relations so that knowledge gaps could be identified in achieving the above strategic objectives of the TPA. Research to date has been found to be insufficient to compile a convincing communication package as described. However, identified knowledge gaps can now be addressed through targeted research. Information derived from targeted research will provide valuable material for education of the end user of turfgrass. Recommendations have been developed, based on the results of this desktop review. It was determined that future research in the field of turf irrigation needs to focus on a number of key factors which directly or indirectly affect the relationship between turfgrass and water use. These factors are: • Climate • Cultivar • Quality • Site use requirements • Establishment and management The overarching recommendation is to develop a strategic plan for turfgrass water relations research based around the five determinants of turf water use listed above. This plan should ensure research under these five categories is integrated into a holistic approach by which the consumer can be guided in species and/or cultivar choices as well as best management practices with respect to turfgrass water relations. Worsening drought cycles and limited supply of water for irrigation were the key factors driving every research project reviewed in this report. Subsidence of the most recent (or current) drought conditions in Australia should not be viewed by the turf industry as a reason to withdraw support or funding for research in this area. Drought conditions, limited domestic water availability and urban water restrictions will return in Australia albeit in 5, 10 or 20 years time and the turf industry has an opportunity to prepare for that time.

Nutrient loss and water quality under extensive grazing in the upper Burdekin river catchment, North Queensland

Increased sediment and nutrient losses resulting from unsustainable grazing management in the Burdekin River catchment are major threats to water quality in the Great Barrier Reef Lagoon. To test the effects of grazing management on soil and nutrient loss, five 1 ha mini-catchments were established in 1999 under different grazing strategies on a sedimentary landscape near Charters Towers. Reference samples were also collected from watercourses in the Burdekin catchment during major flow events.Soil and nutrient loss were relatively low across all grazing strategies due to a combination of good cover, low slope and low rainfall intensities. Total soil loss varied from 3 to 20 kg haˉ¹ per event while losses of N and P ranged from 10 to 1900 g haˉ¹ and from 1 to 71 g haˉ¹ per event respectively. Water quality of runoff was considered moderate across all strategies with relatively low levels of total suspended sediment (range: 8-1409 mg lˉ¹), total N (range: 101-4000 ug lˉ¹) and total P (range: 14-609 ug lˉ¹). However, treatment differences are likely to emerge with time as the impacts of the different grazing strategies on land condition become more apparent.Samples collected opportunistically from rivers and creeks during flow events displayed significantly higher levels of total suspended sediment (range: 10-6010 mg lˉ¹), total N (range: 650-6350 ug lˉ¹) and total P (range: 50-1500 ug lˉ¹) than those collected at the grazing trial. These differences can largely be attributed to variation in slope, geology and cover between the grazing trial and different catchments. In particular, watercourses draining hillier, grano-diorite landscapes with low cover had markedly higher sediment and nutrient loads compared to those draining flatter, sedimentary landscapes.These preliminary data suggest that on relatively flat, sedimentary landscapes, extensive cattle grazing is compatible with achieving water quality targets, provided high levels of ground cover are maintained. In contrast, sediment and nutrient loss under grazing on more erodable land types is cause for serious concern. Long-term empirical research and monitoring will be essential to quantify the impacts of changed land management on water quality in the spatially and temporally variable Burdekin River catchment.

Free convection heat transfer to mercury in vertical annuli

Data on free convection heat transfer to water and mercury are collected using a test rig in vertical annuli of three radii ratios, the walls of which are maintained at uniform temperatures. A theoretical analysis of the boundary layer equations has been attempted using local similarity transformation and double boundary layer approach. Correlations derived from the present theoretical analysis are compared with the analysis and the experimental data available in literature for non-metallic fluids and also with the present experimental data on water and mercury. Generalised correlations are set up for expressing the ratio of heat transferred by convection to the heat transferred by pure conduction and Nusselt's number, in terms of Grashof, Rayleigh and Prandtl numbers, based on the theoretical analysis and the present data on mercury and water. The present generalised correlations agree with the reported and present data for non-metallic fluids and liquid metals with an average deviation of 9% and maximum deviation of ± 13.7%.

Genetic Diversity and Adaptation of Date Palm (Phoenix dactylifera L.)

Acquiring sufficient information on the genetic variation, genetic differentiation, and the ecological and genetic relationships among individuals and populations are essential for establishing guidelines on conservation and utilization of the genetic resources of a species, and more particularly when biotic and abiotic stresses are considered. The aim of this study was to assess the extent and pattern of genetic variation in date palm (Phoenix dacttylifera L) cultivars; the genetic diversity and structure in its populations occurring over geographical ranges; the variation in economically and botanically important traits of it and the variation in its drought adaptive traits, in conservation and utilization context. In this study, the genetic diversity and relationships among selected cultivars from Sudan and Morocco were assessed using microsatellite markers. Microsatellite markers were also used to investigate the genetic diversity within and among populations collected from different geographic locations in Sudan. In a separate investigation, fruits of cultivars selected from Sudan, involved morphological and chemical characterization, and morphological and DNA polymorphism of the mother trees were also investigated. Morphological and photosynthetic adjustments to water stress were studied in the five most important date palm cultivars in Sudan, namely, Gondaila, Barakawi, Bitamoda, Khateeb and Laggai; and the mechanism enhancing photosynthetic gas exchange in date palm under water stress was also investigated. Results showed a significant (p < 0.001, t-test) differentiation between Sudan and Morocco groups of cultivars. However, the major feature of all tested cultivars was the complete lack of clustering and the absence of cultivars representing specific clones. The results indicated high genetic as well as compositional and morphological diversity among cultivars; while, compositional and morphological traits were found to be characteristic features that strongly differentiate cultivars as well as phenotypes. High genetic diversity was observed also in different populations. Slight but significant (p < 0.01, AMOVA) divergence was observed for soft and dry types; however, the genetic divergence among populations was relatively weak. The results showed a complex genetic relationships between some of the tested populations especially when isolation by distance was considered. The results of the study also revealed that date palm cultivars and phenotypes possess specific direct or interaction effects due to water availability on a range of morphological and physiological traits. Soft and dry phenotypes responded differently to different levels of water stress, while the dry phenotype was more sensitive and conservative. The results indicated that date palm has high fixation capacity to photosynthetic CO2 supply with interaction effect to water availability, which can be considered as advantageous when coping with stresses that may arise with climate change. In conclusion, although a large amount of diversity exists among date palm germplasm, the findings in this study show that the role of biological nature of the tree, isolation by distance and environmental effects on structuring date palm genome was highly influenced by human impacts. Identity of date palm cultivars as developed and manipulated by date palm growers, in the absence of scientific breeding programmes, may continue to mainly depend on tree morphology and fruit characters. The pattern of genetic differentiation may cover specific morphological and physiological traits that contribute to adaptive mechanisms in each phenotype. These traits can be considered for further studies related to drought adaptation in date palm.

Foodborne Yersinia : Identification and Molecular Epidemiology of Isolates from Human Infections

Yersinia enterocolitica and Yersinia pseudotuberculosis are among the major enteropathogenic bacteria causing infections in humans in many industrialized countries. In Finland, Y. pseudotuberculosis has caused 10 outbreaks among humans during 1997-2008. Some of these outbreaks have been very extensive involving over 400 cases; mainly children attending schools and day-care. Y. enterocolitica, on the contrary, has caused mainly a large number of sporadic human infections in Finland. Y. pseudotuberculosis is widespread in nature, causing infections in a variety of domestic and wild animals. Foodborne transmission of human infections has long been suspected, however, attempts to trace the pathogen have been unsuccessful before this study that epidemiologically linked Y. pseudotuberculosis to a specific food item. Furthermore, due to modern food distribution systems, foodborne outbreaks usually involve many geographically separate infection clusters difficult to identify as part of the same outbreak. Among pathogenic Y. enterocolitica, the global predominance of one genetically homogeneous type (bioserotype 4/O:3) is a challenge to the development of genetic typing methods discriminatory enough for epidemiological purposes, for example, for tracing back to the sources of infections. Furthermore, the diagnostics of Y. enterocolitica infections is hampered because clinical laboratories easily misidentify some other members of the Yersinia species (Y. enterocolitica–like species) as Y. enterocolitica. This results in misleading information on the prevalence and clinical significance of various Yersinia isolates. The aim of this study was to develop and optimize molecular typing methods to be used in epidemiological investigations of Y. enterocolitica and Y. pseudotuberculosis, particularly in active surveillance and outbreak investigations of Y. pseudotuberculosis isolates. The aim was also to develop a simplified set of phenotypic tests that could be used in routine diagnostic laboratories for the correct identification of Y. enterocolitica and Y. enterocolitica –like species. A PFGE method designed here for typing of Y. pseudotuberculosis was efficient in linking the geographically dispersed and apparently unrelated Y. pseudotuberculosis infections as parts of the same outbreak. It proved to be useful in active laboratory-based surveillance of Y. pseudotuberculosis outbreaks. Throughout the study period, information about the diversity of genotypes among outbreak and non-outbreak related strains of human origin was obtained. Also, to our knowledge, this was the first study to epidemiologically link a Y. pseudotuberculosis outbreak of human illnesses to a specific food item, iceberg lettuce. A novel epidemiological typing method based on the use of a repeated genomic region (YeO:3RS) as a probe was developed for the detection and differentiation between strains of Y. enterocolitica subspecies palearctica. This method was able to increase the discrimination in a set of 106 previously PFGE typed Finnish Y. enterocolitica bioserotype 4/O:3 strains among which two main PFGE genotypes had prevailed. The developed simplified method was a more reliable tool than the commercially available biochemical test kits for differentiation between Y. enterocolitica and Y. enterocolitica –like species. In Finland, the methods developed for Y. enterocolitica and Y. pseudotuberculosis have been used to improve the identification protocols and in subsequent outbreak investigations.

Author responsibilities in improving the quality of peer reviews: A rejoinder to Iivari (2016)

In this rejoinder to Iivari (2016), I discuss authors’ responsibilities in the process of ensuring quality reviews. I argue that one overlooked element in quality peer reviewing is authors’ unconstrained right to submit manuscripts in whatever form or quality they desire. As such, I suggest adding some constraints and offering more freedom to reviewers to maintain viability of the scholarly publication system. I offer three responses to Iivari’s suggestions and add two further suggestions for change.

Growth of Queensland Jatropha gossypifolia biotypes under varying water regimes

Bellyache bush (Jatropha gossypifolia L. (Euphorbiaceae)) is a serious weed of dry tropical regions of northern Australia, with the potential to spread over much of the tropical savannah. It is well adapted to the harsh conditions of the dry tropics, defoliating during the dry season and rapidly producing new leaves with the onset of the wet season. In this study we examined the growth and biomass allocation of the three Queensland biotypes Queensland Green, Queensland Bronze and Queensland Purple) under three water regimes (water-stressed, weekly watering and constant water). Bellyache bush plants have a high capacity to adjust to water stress. The impact of water stress was consistent across the three biotypes. Water stressed plants produced significantly less biomass compared to plants with constant water, increased their biomass allocation to the roots and increased biomass allocation to leaf material. Queensland Purple plants allocated more resources to roots and less to shoots than Queensland Green (Queensland Bronze being intermediate). Queensland Green produced less root biomass than the other two biotypes.

Analysis and prevention of extreme vibration in high speed craft - A research framework

Extreme vibration has been reported for small, high speed craft in the maritime sector, with performance and health threatening effects on boat operators and crew. Musculoskeletal injuries are an enduring problem for high speed craft passengers. Spinal or joint injuries and neurological disorders may occur from repetitive pounding over rough water, continued vibration and single impact events. The risk from whole body vibration (WBV) induced through the small vessels mainly depends on time spent on the craft, which can’t be changed in a military scenario; as well as the number of shocks and jolts, and their magnitude and frequency. In the European Union for example, physical agents directives require all employers to control exposure to a number of physical agents including noise and vibration. The EC Vibration Directive 2002/44/EC then sets out regulations for the control of health and safety risks from the exposure of workers to hand arm vibration (HAV) and WBV in the workplace. Australia has exposure standards relating to WBV, AS 2670.1-2001 – Evaluation of human exposure to whole body vibration. This standard is identical to the ISO 2631-1:1997, Mechanical vibration and shock – Evaluation of human exposure to whole-body vibration. Currently, none of the jurisdictions in Australia have specific regulations for vibration exposures in workplaces. However vibration is mentioned to varying degrees in their general regulations, codes of practice and guidance material. WBV on high speed craft is normally caused by “continuous 'hammering' from short steep seas or wind against tide conditions. Shock on High Speed Craft is usually caused by random impacts. Military organisations need the knowledge to make informed decisions regarding their marine operations, compliance with legislation and potentially harmful health effects, and develop and implement appropriate counter-measures. Marine case studies in the UK such as published MAIB (Marine Accident Investigation Branch) reports show injuries that have occurred in operation, and subsequent MCA (Maritime Coastguard Agency) guidance is provided (MGN 436 (M+F), WHOLE-BODY VIBRATION: Guidance on Mitigating Against the Effects of Shocks and Impacts on Small Vessels. MCA, 2011). This paper proposes a research framework to study the origin, impact and pathways for prevention of WBV in small, high speed craft in a maritime environment.

Kansankirkko, uskonnonvapaus ja valtio : Suomen evankelis-luterilaisen kirkon kirkolliskokouksen tulkinta uskonnonvapaudesta 1963-2003

A national church, freedom of religion, and the state The interpretation of freedom of religion formulated by the Synod of the Evangelical Lutheran Church of Finland in reference to the relationship between the Church and the state from 1963 to 2003 This paper discusses the interpretation of freedom of religion formulated by the Synod of the Evangelical Lutheran Church of Finland during the years 1963-2003. The effect of these formulations and decisions made by the Synod on the relationship between the Church and the state is also discussed as the relationship has been a central issue in the debate about freedom of religion in Finland. Active co-operation with the state caused a dispute in the Church during this period. Another cause for concern for the Synod, a strong defender of the national church, was the weakening position of the Church in a society undergoing many changes. As the Synod of 1963 discussed the status of the Church, the Church began to reflect upon its identity as a national church, and to evaluate freedom of religion in the country, as well as the relationship between the Church and the state. Some of the radicals of the 1960s and 1970s presented the Church as an obstacle to freedom of religion. The Synod was keen to emphasize that, in accordance with international agreements on human rights, freedom of religion means the freedom to have and follow a religion, and also that freedom of religion was a right of the majority in Finnish society. As an active guardian of the rights of its members, the Synod defended such issues as the teaching of religion in schools. Throughout the dispute, the Church focused on its right to act freely and, according to its identity, to express spirituality in the society. At the end of the 1960s, several efforts to reform the law on the freedom of religion and the relationship between the Church and the state gained favour in the Synod. These formulations of the Church were the basis for the work of a parliamentary committee in the 1970s, but no significant changes resulted. Instead, freedom of religion in Finland was judged to be fairly good. The committee paper did, however, lead to preparations for greater independence of the Church. The Synod at the time chose to react to the changes presented to it, but it was not before the 1990s that the Synod became an active force of reform in these matters. Though the Synod, particularly from the 1970s onwards, began clearly to favour the improvement of the position of other religious communities in Finland, it felt it had reason to be cautious as each church and religious community had the freedom to decide individually its relationship with the state. Any changes that would have weakened the position of the Church in Finnish society were met with disapproval in the Synod. Even though some theological concerns regarding the national identity of the Church were raised, the Synod emphasized issues of church policy. Keen to preserve and protect its legal status in society, the Synod judged that this status supported the freedom of action enjoyed by the Church as well as the freedom of religion.

Kansankirkko, uskonnonvapaus ja valtio : Suomen evankelis-luterilaisen kirkon kirkolliskokouksen tulkinta uskonnonvapaudesta 1963-2003

A national church, freedom of religion, and the state The interpretation of freedom of religion formulated by the Synod of the Evangelical Lutheran Church of Finland in reference to the relationship between the Church and the state from 1963 to 2003 This paper discusses the interpretation of freedom of religion formulated by the Synod of the Evangelical Lutheran Church of Finland during the years 1963-2003. The effect of these formulations and decisions made by the Synod on the relationship between the Church and the state is also discussed as the relationship has been a central issue in the debate about freedom of religion in Finland. Active co-operation with the state caused a dispute in the Church during this period. Another cause for concern for the Synod, a strong defender of the national church, was the weakening position of the Church in a society undergoing many changes. As the Synod of 1963 discussed the status of the Church, the Church began to reflect upon its identity as a national church, and to evaluate freedom of religion in the country, as well as the relationship between the Church and the state. Some of the radicals of the 1960s and 1970s presented the Church as an obstacle to freedom of religion. The Synod was keen to emphasize that, in accordance with international agreements on human rights, freedom of religion means the freedom to have and follow a religion, and also that freedom of religion was a right of the majority in Finnish society. As an active guardian of the rights of its members, the Synod defended such issues as the teaching of religion in schools. Throughout the dispute, the Church focused on its right to act freely and, according to its identity, to express spirituality in the society. At the end of the 1960s, several efforts to reform the law on the freedom of religion and the relationship between the Church and the state gained favour in the Synod. These formulations of the Church were the basis for the work of a parliamentary committee in the 1970s, but no significant changes resulted. Instead, freedom of religion in Finland was judged to be fairly good. The committee paper did, however, lead to preparations for greater independence of the Church. The Synod at the time chose to react to the changes presented to it, but it was not before the 1990s that the Synod became an active force of reform in these matters. Though the Synod, particularly from the 1970s onwards, began clearly to favour the improvement of the position of other religious communities in Finland, it felt it had reason to be cautious as each church and religious community had the freedom to decide individually its relationship with the state. Any changes that would have weakened the position of the Church in Finnish society were met with disapproval in the Synod. Even though some theological concerns regarding the national identity of the Church were raised, the Synod emphasized issues of church policy. Keen to preserve and protect its legal status in society, the Synod judged that this status supported the freedom of action enjoyed by the Church as well as the freedom of religion.

Essays in Philosophical Moral Psychology

The dissertation consists of four essays and a comprehensive introduction that discusses the topics, methods, and most prominent theories of philosophical moral psychology. I distinguish three main questions: What are the essential features of moral thinking? What are the psychological conditions of moral responsibility? And finally, what are the consequences of empirical facts about human nature to normative ethics? Each of the three last articles focuses on one of these issues. The first essay and part of the introduction are dedicated to methodological questions, in particular the relationship between empirical (social) psychology and philosophy. I reject recent attempts to understand the nature of morality on the basis of empirical research. One characteristic feature of moral thinking is its practical clout: if we regard an action as morally wrong, we either refrain from doing it even against our desires and interests, or else feel shame or guilt. Moral views seem to have a conceptual connection to motivation and emotions – roughly speaking, we can’t conceive of someone genuinely disapproving an action, but nonetheless doing it without any inner motivational conflict or regret. This conceptual thesis in moral psychology is called (judgment) internalism. It implies, among other things, that psychopaths cannot make moral judgments to the extent that they are incapable of corresponding motivation and emotion, even if they might say largely the words we would expect. Is internalism true? Recently, there has been an explosion of interest in so-called experimental philosophy, which is a methodological view according to which claims about conceptual truths that appeal to our intuitions should be tested by way of surveys presented to ordinary language users. One experimental result is that the majority of people are willing to grant that psychopaths make moral judgments, which challenges internalism. In the first article, ‘The Rise and Fall of Experimental Philosophy’, I argue that these results pose no real threat to internalism, since experimental philosophy is based on a too simple conception of the relationship between language use and concepts. Only the reactions of competent users in pragmatically neutral and otherwise conducive circumstances yield evidence about conceptual truths, and such robust intuitions remain inaccessible to surveys for reasons of principle. The epistemology of folk concepts must still be based on Socratic dialogue and critical reflection, whose character and authority I discuss at the end of the paper. The internal connection between moral judgment and motivation led many metaethicists in the past century to believe along Humean lines that judgment itself consists in a pro-attitude rather than a belief. This expressivist view, as it is called these days, has far-reaching consequences in metaethics. In the second essay I argue that perhaps the most sophisticated form of contemporary expressivism, Allan Gibbard’s norm-expressivism, according to which moral judgments are decisions or contingency plans, is implausible from the perspective of the theory of action. In certain circumstances it is possible to think that something is morally required of one without deciding to do so. Morality is not a matter of the will. Instead, I sketch on the basis of Robert Brandom’s inferentialist semantics a weak form of judgment internalism, according to which the content of moral judgment is determined by a commitment to a particular kind of practical reasoning. The last two essays in the dissertation emphasize the role of mutual recognition in the development and maintenance of responsible and autonomous moral agency. I defend a compatibilist view of autonomy, according to which agents who are unable to recognize right and wrong or act accordingly are not responsible for their actions – it is not fair to praise or blame them, since they lacked the relevant capacity to do otherwise. Conversely, autonomy demands an ability to recognize reasons and act on them. But as a long tradition in German moral philosophy whose best-known contemporary representative is Axel Honneth has it, both being aware of reasons and acting on them requires also the right sort of higher-order attitudes toward the self. Without self-respect and self-confidence we remain at the mercy of external pressures, even if we have the necessary normative competence. These attitudes toward the self, in turn, are formed through mutual recognition – we value ourselves when those who we value value us. Thus, standing in the right sort of relations of recognition is indirectly necessary for autonomy and moral responsibility. Recognition and valuing are concretely manifest in actions and institutions, whose practices make possible participation on an equal footing. Seeing this opens the way for a kind of normative social criticism that is grounded in the value of freedom and automomy, but is not limited to defending negative rights. It thus offers a new way to bridge the gap between liberalism and communitarianism.

Curious hybrids: Creating 'not-quite' beings to explore possible childhoods

Communications between adults and young children can expose different ideas and opinions. Adults and children have different capacities to speak, these discursive spaces can become filled with assumptions, stereotyping and conventional thinking about power and agency. If communication shifts away from the purely discursive, what might be exposed about the explorations, investigations and fantasies adults and children indulge in? Some time ago my young daughter obsessively drew hybrid beings. Created from mixtures of animal, object, human and creature forms, these beings, which are ‘not-quite’, are becoming, able to transform via myriad mutations. We agreed to collaborate and draw additional hybrid beings to experiment with becoming-other through complex entanglements of forms, to complicate, morph and (trans)form from our human selves to hybrid others. The ‘not- quite-ness’ of our monstrous hybrids subvert the conventions of ‘being’ and prompt contemplations about childhood subjectivities, identities, conventionalities and actively interrogate the assumptive knowledges and subjectifications that are held about young children in early childhood professional and academic systems.

Structural and Functional Studies on Trimeric Autotransporters

Trimeric autotransporters are a family of secreted outer membrane proteins in Gram-negative bacteria. These obligate homotrimeric proteins share a conserved C-terminal region, termed the translocation unit. This domain consists of an integral membrane β-barrel anchor and associated α-helices which pass through the pore of the barrel. The α-helices link to the extracellular portion of the protein, the passenger domain. Autotransportation refers to the way in which the passenger domain is secreted into the extracellular space. It appears that the translocation unit mediates the transport of the passenger domain across the outer membrane, and no external factors, such as ATP, ion gradients nor other proteins, are required. The passenger domain of autotransporters contains the specific activities of each protein. These are usually related to virulence. In trimeric autotransporters, the main function of the proteins is to act as adhesins. One such protein is the Yersinia adhesin YadA, found in enteropathogenic species of Yersinia. The main activity of YadA from Y. enterocolitica is to bind collagen, and it also mediates adhesion to other molecules of the extracellular matrix. In addition, YadA is involved in serum resistance, phagocytosis resistance, binding to epithelial cells and autoagglutination. YadA is an essential virulence factor of Y. enterocolitica, and removal of this protein from the bacteria leads to avirulence. In this study, I investigated the YadA-collagen interaction by studying the binding of YadA to collagen-mimicking peptides by several biochemical and biophysical methods. YadA bound as tightly to the triple-helical model peptide (Pro-Hyp-Gly)10 as to native collagen type I. However, YadA failed to bind a similar peptide that does not form a collagenous triple helix. As (Pro-Hyp-Gly)10 does not contain a specific sequence, we concluded that a triple-helical conformation is necessary for YadA binding, but no specific sequence is required. To further investigate binding determinants for YadA in collagens, I examined the binding of YadA to a library of collagen-mimicking peptides that span the entire triple-helical sequences of human collagens type II and type III. YadA bound promiscuously to many but not all peptides, indicating that a triple-helical conformation alone is not sufficient for binding. The high-binding peptides did not share a clear binding motif, but these peptides were rich in hydroxyproline residues and contained a low number of charged residues. YadA thus binds collagens without sequence specificity. This strategy of promiscuous binding may be advantageous for pathogenic bacteria. The Eib proteins from Escherichia coli are immunoglobulin (Ig)-binding homologues of YadA. I showed conclusively that recombinant EibA, EibC, EibD and EibF bind to IgG Fc. I crystallised a fragment of the passenger domain of EibD, which binds IgA in addition to IgG. The structure has a YadA-like head domain and an extended coiled-coil stalk. The top half of the coiled-coil is right-handed with hendecad periodicity, whereas the lower half is a canonical left-handed coiled-coil. At the transition from right- to left-handedness, a small β-sheet protrudes from each monomer. I was able to map the binding regions for IgG and IgA using truncations and site-directed mutagenesis to the coiled-coil stalk and identified residues critical for Ig binding.

The high- and low-activity forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.