A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.

Agroforestry systems for sustainable livelihoods and improved land management in the East Usambara Mountains, Tanzania

Forest destruction for agriculture continues to be a major threat to the rich biological diversity in the East Usambara Mountains in the north-eastern corner of Tanzania. The highest ratio of endemic plant and animal species found on 100 km2 anywhere in the world is depending on the remaining natural forests. Forests are vitally important for the local population in many different ways, and nationally they are an important source of water and hydroelectricity. The soils, of low fertility and mostly acidic Ferrasols, mainly have the nutrients in the topsoil. After clear-cutting, the soils soon become poor when the topsoil is eroded. High-value cardamom is nowadays unsustainably cultivated in the natural forests of the East Usambaras. The general aim was to study the possibilities to develop new profitable and sustainable agroforestry systems for the benefit of the local people that could contribute to relieving the pressure on the remaining natural forests in the East Usambara Mountains. Results from a spice crop agroforestry trial, established in cooperation with a local farmer, showed a clear advantage of intercropping cardamom (Elettaria cardamomum) and black pepper (Piper nigrum) with trees, especially with Grevillea robusta. The nitrogen fixing tree species Gliricidia sepium also improved the nitrogen and organic matter content of the soil over levels found in the natural forest. With improved agroforestry methods for spice production the households generated as much as13 times the net income obtained with traditional forest cultivation practices. There are thus sustainable and profitable ways to cultivate spices as cash crops in well-managed homegardens. However, the farmers need stable markets, access to credit and comprehensive extension services. The soil fertility depletion should be reversed with organic manure application and an enabling policy environment for the smallholder-farming sector. Strong farmers organisations and equal rights to resources and decision-making are needed. Organic spices have an increasing demand, and their export would be profitable for these farmers. What is, however, most needed for a change is a political will of a government that understands the importance of agricultural and forestry development for poverty reduction.

Ecology, population genetics and conservation of the African violet

The ongoing rapid fragmentation of tropical forests is a major threat to global biodiversity. This is because many of the tropical forests are so-called biodiversity 'hotspots', areas that host exceptional species richness and concentrations of endemic species. Forest fragmentation has negative ecological and genetic consequences for plant survival. Proposed reasons for plant species' loss in forest fragments are, e.g., abiotic edge effects, altered species interactions, increased genetic drift, and inbreeding depression. To be able to conserve plants in forest fragments, the ecological and genetic processes that threaten the species have to be understood. That is possible only after obtaining adequate information on their biology, including taxonomy, life history, reproduction, and spatial and genetic structure of the populations. In this research, I focused on the African violet (genus Saintpaulia), a little-studied conservation flagship from the Eastern Arc Mountains and Coastal Forests hotspot of Tanzania and Kenya. The main objective of the research was to increase understanding of the life history, ecology and population genetics of Saintpaulia that is needed for the design of appropriate conservation measures. A further aim was to provide population-level insights into the difficult taxonomy of Saintpaulia. Ecological field work was conducted in a relatively little fragmented protected forest in the Amani Nature Reserve in the East Usambara Mountains, in northeastern Tanzania, complemented by population genetic laboratory work and ecological experiments in Helsinki, Finland. All components of the research were conducted with Saintpaulia ionantha ssp. grotei, which forms a taxonomically controversial population complex in the study area. My results suggest that Saintpaulia has good reproductive performance in forests with low disturbance levels in the East Usambara Mountains. Another important finding was that seed production depends on sufficient pollinator service. The availability of pollinators should thus be considered in the in situ management of threatened populations. Dynamic population stage structures were observed suggesting that the studied populations are demographically viable. High mortality of seedlings and juveniles was observed during the dry season but this was compensated by ample recruitment of new seedlings after the rainy season. Reduced tree canopy closure and substrate quality are likely to exacerbate seedling and juvenile mortality, and, therefore, forest fragmentation and disturbance are serious threats to the regeneration of Saintpaulia. Restoration of sufficient shade to enhance seedling establishment is an important conservation measure in populations located in disturbed habitats. Long-term demographic monitoring, which enables the forecasting of a population s future, is also recommended in disturbed habitats. High genetic diversities were observed in the populations, which suggest that they possess the variation that is needed for evolutionary responses in a changing environment. Thus, genetic management of the studied populations does not seem necessary as long as the habitats remain favourable for Saintpaulia. The observed high levels of inbreeding in some of the populations, and the reduced fitness of the inbred progeny compared to the outbred progeny, as revealed by the hand-pollination experiment, indicate that inbreeding and inbreeding depression are potential mechanisms contributing to the extinction of Saintpaulia populations. The relatively weak genetic divergence of the three different morphotypes of Saintpaulia ionantha ssp. grotei lend support to the hypothesis that the populations in the Usambara/lowlands region represent a segregating metapopulation (or metapopulations), where subpopulations are adapting to their particular environments. The partial genetic and phenological integrity, and the distinct trailing habit of the morphotype 'grotei' would, however, justify its placement in a taxonomic rank of its own, perhaps in a subspecific rank.

Effect of water vapour stabilization of raw materials on the quality of the inhalation product

Generation of raw materials for dry powder inhalers by different size reduction methods can be expected to influence physical and chemical properties of the powders. This can cause differences in particle size, size distribution, shape, crystalline properties, surface texture and energy. These physical properties of powders influence the behaviour of particles before and after inhalation. Materials with an amorphous surface have different surface energy compared to materials with crystalline surface. This can affect the adhesion and cohesion of particles. Changes in the surface nature of the drug particles results in a change in product performance. By stabilization of the raw materials the amorphous surfaces are converted into crystalline surfaces. The primary aim of the study was to investigate the influence of the surface properties of the inhalation particles on the quality of the product. The quality of the inhalation product is evaluated by measuring the fine particle dose (FPD). FDP is the total dose of particles with aerodynamic diameters smaller than 5,0 μm. The secondary aim of this study was to achieve the target level of the FPD and the stability of the FPD. This study was also used to evaluate the importance of the stabilization of the inhalation powders. The study included manufacturing and analysing drug substance 200 μg/dose inhalation powder batches using non-stabilized or stabilized raw materials. The inhaler formulation consisted of micronized drug substance, lactose <100μm and micronized lactose <10μm. The inhaler device was Easyhaler®. Stabilization of the raw materials was done in different relative humidity, temperature and time. Surface properties of the raw materials were studied by dynamic vapour sorption, scanning electron microscopy and three-point nitrogen adsorption technique. Particle size was studied by laser diffraction particle size analyzer. Aerodynamic particle size distribution from inhalers was measured by new generation impactor. Stabilization of all three raw materials was successful. A clear difference between nonstabilized and stabilized raw materials was achieved for drug substance and lactose <10μm. However for lactose <100μm the difference wasn’t as clear as wanted. The surface of the non-stabilized drug substance was more irregular and the particles had more roughness on the surface compared to the stabilized drug substances particles surface. The surface of the stabilized drug particles was more regular and smoother than non-stabilized. Even though a good difference between stabilized and non-stabilized raw materials was achieved, a clear evidence of the effect of the surface properties of the inhalation particles on the quality of the product was not observed. Stabilization of the raw materials didn’t lead to a higher FPD. Possible explanations for the unexpected result might be too rough conditions in the stabilization of the drug substance or smaller than wanted difference in the degree of stabilization of the main component of the product <100μm. Despite positive effects on the quality of the product were not seen there appears to be some evidence that stabilized drug substance results in smaller particle size of dry powder inhalers.