Understanding solid-state transformations during dehydration: new insights using vibrational spectroscopy and multivariate modelling

Many active pharmaceutical ingredients (APIs) have both anhydrate and hydrate forms. Due to the different physicochemical properties of solid forms, the changes in solid-state may result in therapeutic, pharmaceutical, legal and commercial problems. In order to obtain good solid dosage form quality and performance, there is a constant need to understand and control these phase transitions during manufacturing and storage. Thus it is important to detect and also quantify the possible transitions between the different forms. In recent years, vibrational spectroscopy has become an increasingly popular tool to characterise the solid-state forms and their phase transitions. It offers several advantages over other characterisation techniques including an ability to obtain molecular level information, minimal sample preparation, and the possibility of monitoring changes non-destructively in-line. Dehydration is the phase transition of hydrates which is frequently encountered during the dosage form production and storage. The aim of the present thesis was to investigate the dehydration behaviour of diverse pharmaceutical hydrates by near infrared (NIR), Raman and terahertz pulsed spectroscopic (TPS) monitoring together with multivariate data analysis. The goal was to reveal new perspectives for investigation of the dehydration at the molecular level. Solid-state transformations were monitored during dehydration of diverse hydrates on hot-stage. The results obtained from qualitative experiments were used to develop a method and perform the quantification of the solid-state forms during process induced dehydration in a fluidised bed dryer. Both in situ and in-line process monitoring and quantification was performed. This thesis demonstrated the utility of vibrational spectroscopy techniques and multivariate modelling to monitor and investigate dehydration behaviour in situ and during fluidised bed drying. All three spectroscopic methods proved complementary in the study of dehydration. NIR spectroscopy models could quantify the solid-state forms in the binary system, but were unable to quantify all the forms in the quaternary system. Raman spectroscopy models on the other hand could quantify all four solid-state forms that appeared upon isothermal dehydration. The speed of spectroscopic methods makes them applicable for monitoring dehydration and the quantification of multiple forms was performed during phase transition. Thus the solid-state structure information at the molecular level was directly obtained. TPS detected the intermolecular phonon modes and Raman spectroscopy detected mostly the changes in intramolecular vibrations. Both techniques revealed information about the crystal structure changes. NIR spectroscopy, on the other hand was more sensitive to water content and hydrogen bonding environment of water molecules. This study provides a basis for real time process monitoring using vibrational spectroscopy during pharmaceutical manufacturing.

Electron cryo-microscopy studies of bacteriophage phi8 and archaeal virus SH1

Symmetry is a key principle in viral structures, especially the protein capsid shells. However, symmetry mismatches are very common, and often correlate with dynamic functionality of biological significance. The three-dimensional structures of two isometric viruses, bacteriophage phi8 and the archaeal virus SH1 were reconstructed using electron cryo-microscopy. Two image reconstruction methods were used: the classical icosahedral method yielded high resolution models for the symmetrical parts of the structures, and a novel asymmetric in-situ reconstruction method allowed us to resolve the symmetry mismatches at the vertices of the viruses. Evidence was found that the hexameric packaging enzyme at the vertices of phi8 does not rotate relative to the capsid. The large two-fold symmetric spikes of SH1 were found not to be responsible for infectivity. Both virus structures provided insight into the evolution of viruses. Comparison of the phi8 polymerase complex capsid with those of phi6 and other dsRNA viruses suggests that the quaternary structure in dsRNA bacteriophages differs from other dsRNA viruses. SH1 is unusual because there are two major types of capsomers building up the capsid, both of which seem to be composed mainly of single beta-barrels perpendicular to the capsid surface. This indicates that the beta-barrel may be ancestral to the double beta-barrel fold.

Interatomic potentials for fusion reactor material simulations

Fusion energy is a clean and safe solution for the intricate question of how to produce non-polluting and sustainable energy for the constantly growing population. The fusion process does not result in any harmful waste or green-house gases, since small amounts of helium is the only bi-product that is produced when using the hydrogen isotopes deuterium and tritium as fuel. Moreover, deuterium is abundant in seawater and tritium can be bred from lithium, a common metal in the Earth's crust, rendering the fuel reservoirs practically bottomless. Due to its enormous mass, the Sun has been able to utilize fusion as its main energy source ever since it was born. But here on Earth, we must find other means to achieve the same. Inertial fusion involving powerful lasers and thermonuclear fusion employing extreme temperatures are examples of successful methods. However, these have yet to produce more energy than they consume. In thermonuclear fusion, the fuel is held inside a tokamak, which is a doughnut-shaped chamber with strong magnets wrapped around it. Once the fuel is heated up, it is controlled with the help of these magnets, since the required temperatures (over 100 million degrees C) will separate the electrons from the nuclei, forming a plasma. Once the fusion reactions occur, excess binding energy is released as energetic neutrons, which are absorbed in water in order to produce steam that runs turbines. Keeping the power losses from the plasma low, thus allowing for a high number of reactions, is a challenge. Another challenge is related to the reactor materials, since the confinement of the plasma particles is not perfect, resulting in particle bombardment of the reactor walls and structures. Material erosion and activation as well as plasma contamination are expected. Adding to this, the high energy neutrons will cause radiation damage in the materials, causing, for instance, swelling and embrittlement. In this thesis, the behaviour of a material situated in a fusion reactor was studied using molecular dynamics simulations. Simulations of processes in the next generation fusion reactor ITER include the reactor materials beryllium, carbon and tungsten as well as the plasma hydrogen isotopes. This means that interaction models, {\it i.e. interatomic potentials}, for this complicated quaternary system are needed. The task of finding such potentials is nonetheless nearly at its end, since models for the beryllium-carbon-hydrogen interactions were constructed in this thesis and as a continuation of that work, a beryllium-tungsten model is under development. These potentials are combinable with the earlier tungsten-carbon-hydrogen ones. The potentials were used to explain the chemical sputtering of beryllium due to deuterium plasma exposure. During experiments, a large fraction of the sputtered beryllium atoms were observed to be released as BeD molecules, and the simulations identified the swift chemical sputtering mechanism, previously not believed to be important in metals, as the underlying mechanism. Radiation damage in the reactor structural materials vanadium, iron and iron chromium, as well as in the wall material tungsten and the mixed alloy tungsten carbide, was also studied in this thesis. Interatomic potentials for vanadium, tungsten and iron were modified to be better suited for simulating collision cascades that are formed during particle irradiation, and the potential features affecting the resulting primary damage were identified. Including the often neglected electronic effects in the simulations was also shown to have an impact on the damage. With proper tuning of the electron-phonon interaction strength, experimentally measured quantities related to ion-beam mixing in iron could be reproduced. The damage in tungsten carbide alloys showed elemental asymmetry, as the major part of the damage consisted of carbon defects. On the other hand, modelling the damage in the iron chromium alloy, essentially representing steel, showed that small additions of chromium do not noticeably affect the primary damage in iron. Since a complete assessment of the response of a material in a future full-scale fusion reactor is not achievable using only experimental techniques, molecular dynamics simulations are of vital help. This thesis has not only provided insight into complicated reactor processes and improved current methods, but also offered tools for further simulations. It is therefore an important step towards making fusion energy more than a future goal.

Late Pleistocene and Holocene environmental evolution of the Murchisonfjorden area, Nordaustlandet, Svalbard

The purpose of this study was to establish the palaeoenvironmental conditions during the late Quaternary in Murchisonfjorden, Nordaustlandet, based on foraminiferal assemblage compositions, and to determine the onset and termination of the Weichselian glaciations. The foraminiferal assemblage compositions were studied in marine sediments from three different archives, from sections next to the present shoreline in the Bay of Isvika, from a core in the Bay of Isvika and from a core from Lake Einstaken. OSL and AMS 14C age determinations were performed on samples from the three archives, and the results show deposition of marine sediments during ice-free periods of the Early Weichselian, the Middle Weichselian and the Late Weichselian, as well as during the Holocene in the investigated area. Marine sediments from the Early and Middle Weichselian were sampled from isostatically uplifted sections along the present shoreline.Sediments from the transition from the Late Weichselian to early Holocene time intervals were found in the bottom of the core from Lake Einstaken. Holocene sediments were investigated in the sections and in the core from the Bay of Isvika. The marine sediments from the sections are comprised of five benthic foraminiferal assemblages. The Early Weichselian is represented by two foraminiferal assemblages, the Middle Weichselian, the early and the late Holocene each by one. All five foraminiferal assemblages were deposited in glacier-distal shallow-water environments, which had a connection to the open ocean. Changes in the composition of the assemblages can be ascribed to differences in the bottom-water currents and changes in the salinity. The Middle Weichselian assemblage is of special importance, because it is the first foraminiferal assemblage to be described from this time interval from Svalbard. Four benthic foraminiferal assemblages were deposited shortly before the marine to lacustrine transition at the boundary between the Late Weichselian and Holocene in Lake Einstaken. The foraminiferal assemblages show a change from a high-arctic, normal marine shallow-water environment to an even shallower environment with highly fluctuating salinity. The analyses of the core from 100 m water depth in the Bay of Isvika resulted in the determination of four foraminiferal assemblages. These indicated changes from a glacier-proximal environment during deglaciation, to a more glacier-distal environment during the Early Holocene. This was followed by a period with a marked change to a considerably cooler environment and finally to a closed fjord environment in the middle and late Holocene times. Additional sedimentological analyses of the marine and glacially derived sediments from the uplifted sections, as well as observations of multiple striae on the bedrock, observations of deeply weathered bedrock and findings of tills interlayered with marine sediments complete the investigations in the study area. They indicate weak glacial erosion in the study area. It can be concluded that marine deposition occurred in the investigated area during three time intervals in the Weichselian and during most of the Holocene. The foraminiferal assemblages in the Holocene are characterized by a transition from glacier-proximal to glacier-distal faunas. The palaeogeographical change from an open fjord to a closed fjord environment is a result of the isostatic uplift of the area after the LGM and is clearly reflected in the foraminiferal assemblages. Another influencing factor on the foraminiferal assemblage composition are changes in the inflow of warmer Atlantic waters to the study area.

Phylogeography and demographic history of Lacerta lepida in the Iberian Peninsula

The Iberian Peninsula is recognized as an important refugial area for species survival and diversification during the climatic cycles of the Quaternary. Recent phylogeographic studies have revealed Iberia as a complex of multiple refugia. However, most of these studies have focused either on species with narrow distributions within the region or species groups that, although widely distributed, generally have a genetic structure that relates to pre-Quaternary cladogenetic events. In this study we undertake a detailed phylogeographic analysis of the lizard species, Lacerta lepida, whose distribution encompasses the entire Iberian Peninsula. We attempt to identify refugial areas, recolonization routes, zones of secondary contact and date demographic events within this species. Results support the existence of 6 evolutionary lineages (phylogroups) with a strong association between genetic variation and geography, suggesting a history of allopatric divergence in different refugia. Diversification within phylogroups is concordant with the onset of the Pleistocene climatic oscillations. The southern regions of several phylogroups show a high incidence of ancestral alleles in contrast with high incidence of recently derived alleles in northern regions. All phylogroups show signs of recent demographic and spatial expansions. We have further identified several zones of secondary contact, with divergent mitochondrial haplotypes occurring in narrow zones of sympatry. The concordant patterns of spatial and demographic expansions detected within phylogroups, together with the high incidence of ancestral haplotypes in southern regions of several phylogroups, suggests a pattern of contraction of populations into southern refugia during adverse climatic conditions from which subsequent northern expansions occurred. This study supports the emergent pattern of multiple refugia within Iberia but adds to it by identifying a pattern of refugia coincident with the southern distribution limits of individual evolutionary lineages. These areas are important in terms of long-term species persistence and therefore important areas for conservation.