Characterization of New Viruses from Hypersaline Environments

Viruses of Archaea are the least studied group of viruses. Fewer than 50 archaeal viruses have been reported which constitutes less than one percent of all the isolated prokaryotic viruses. Only about one third of the isolated archaeal viruses infect halophiles. The diversity of haloviruses, virus ecology in highly saline environments and the interactions of haloviruses with their hosts have been little studied. The exiguous knowledge available on halophilic systems is not only due to inadequate sampling but also reflects the extra challenge highly saline systems set on biochemical studies. In this study six new haloviruses were isolated and characterized. Viruses included four archaeal viruses and two bacteriophages. All of the other isolates exhibited head-tail morphology, except SH1 which was the first tailless icosahedral virus isolated from a high salt environment. Production and purification procedures were set up for all of these viruses and they were subjected to stability determinations. Archaeal virus SH1 was studied in more detail. Biochemical studies revealed an internal membrane underneath the protein capsid and a linear dsDNA genome. The overall structure of SH1 resembles phages PRD1, PM2 and Bam35 as well as an archaeal virus STIV. SH1 possesses about 15 structural proteins that form complexes under non-reducing conditions. Quantitative dissociation provided information about the positions of these proteins in the virion. The life cycle of SH1 was also studied. This lytic virus infects Haloarcula hispanica. Adsorption to the host cells is fairly inefficient and the life cycle rather long. Finally, virus responses in a variety of ionic conditions were studied. It was discovered that all of the studied viruses from low salt, marine and high salt environments tolerated larger range of salinities than their bacterial or archaeal hosts. The adsorption efficiency was not determined by the natural environment of a virus. Even though viruses with the slowest binding kinetics were among the haloviruses, fast binders were observed in viruses from all environments. When the salinity was altered, the virus adsorption responses were diverse. Four different behavioral patterns were observed: virus binding increased or decreased in increasing salinity, adsorption maximum was at a particular salt concentration or the salinity did not affect the binding. The way the virus binding was affected did not correlate with the environment, virus morphology or the organism the virus infects.

New approaches to improve the cord blood unit hematopoietic progenitor cell content

Cord blood is a well-established alternative to bone marrow and peripheral blood stem cell transplantation. To this day, over 400 000 unrelated donor cord blood units have been stored in cord blood banks worldwide. To enable successful cord blood transplantation, recent efforts have been focused on finding ways to increase the hematopoietic progenitor cell content of cord blood units. In this study, factors that may improve the selection and quality of cord blood collections for banking were identified. In 167 consecutive cord blood units collected from healthy full-term neonates and processed at a national cord blood bank, mean platelet volume (MPV) correlated with the numbers of cord blood unit hematopoietic progenitors (CD34+ cells and colony-forming units); this is a novel finding. Mean platelet volume can be thought to represent general hematopoietic activity, as newly formed platelets have been reported to be large. Stress during delivery is hypothesized to lead to the mobilization of hematopoietic progenitor cells through cytokine stimulation. Accordingly, low-normal umbilical arterial pH, thought to be associated with perinatal stress, correlated with high cord blood unit CD34+ cell and colony-forming unit numbers. The associations were closer in vaginal deliveries than in Cesarean sections. Vaginal delivery entails specific physiological changes, which may also affect the hematopoietic system. Thus, different factors may predict cord blood hematopoietic progenitor cell numbers in the two modes of delivery. Theoretical models were created to enable the use of platelet characteristics (mean platelet volume) and perinatal factors (umbilical arterial pH and placental weight) in the selection of cord blood collections with high hematopoietic progenitor cell counts. These observations could thus be implemented as a part of the evaluation of cord blood collections for banking. The quality of cord blood units has been the focus of several recent studies. However, hemostasis activation during cord blood collection is scarcely evaluated in cord blood banks. In this study, hemostasis activation was assessed with prothrombin activation fragment 1+2 (F1+2), a direct indicator of thrombin generation, and platelet factor 4 (PF4), indicating platelet activation. Altogether three sample series were collected during the set-up of the cord blood bank as well as after changes in personnel and collection equipment. The activation decreased from the first to the subsequent series, which were collected with the bank fully in operation and following international standards, and was at a level similar to that previously reported for healthy neonates. As hemostasis activation may have unwanted effects on cord blood cell contents, it should be minimized. The assessment of hemostasis activation could be implemented as a part of process control in cord blood banks. Culture assays provide information about the hematopoietic potential of the cord blood unit. In processed cord blood units prior to freezing, megakaryocytic colony growth was evaluated in semisolid cultures with a novel scoring system. Three investigators analyzed the colony assays, and the scores were highly concordant. With such scoring systems, the growth potential of various cord blood cell lineages can be assessed. In addition, erythroid cells were observed in liquid cultures of cryostored and thawed, unseparated cord blood units without exogenous erythropoietin. This was hypothesized to be due to the erythropoietic effect of thrombopoietin, endogenous erythropoietin production, and diverse cell-cell interactions in the culture. This observation underscores the complex interactions of cytokines and supporting cells in the heterogeneous cell population of the thawed cord blood unit.

Experiments on homogeneous nucleation and physicochemical properties related to atmospheric new particle formation

Aerosol particles play a role in the earth ecosystem and affect human health. A significant pathway of producing aerosol particles in the atmosphere is new particle formation, where condensable vapours nucleate and these newly formed clusters grow by condensation and coagulation. However, this phenomenon is still not fully understood. This thesis brings an insight to new particle formation from an experimental point of view. Laboratory experiments were conducted both on the nucleation process and physicochemical properties related to new particle formation. Nucleation rate measurements are used to test nucleation theories. These theories, in turn, are used to predict nucleation rates in atmospheric conditions. However, the nucleation rate measurements have proven quite difficult to conduct, as different devices can yield nucleation rates with differences of several orders of magnitude for the same substances. In this thesis, work has been done to have a greater understanding in nucleation measurements, especially those conducted in a laminar flow diffusion chamber. Systematic studies of nucleation were also made for future verification of nucleation theories. Surface tensions and densities of substances related to atmospheric new particle formation were measured. Ternary sulphuric acid + ammonia + water is a proposed candidate to participate in atmospheric nucleation. Surface tensions of an alternative candidate to nucleate in boreal forest areas, sulphuric acid + dimethylamine + water, were also measured. Binary compounds, consisting of organic acids + water are possible candidates to participate in the early growth of freshly nucleated particles. All the measured surface tensions and densities were fitted with equations, thermodynamically consistent if possible, to be easily applied to atmospheric model calculations of nucleation and subsequent evolution of particle size.