Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection: From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.

Problems of Proto-Slavic Historical Nominal Morphology : On the Basis of Old Church Slavic

In this study I offer a diachronic solution for a number of difficult inflectional endings in Old Church Slavic nominal declensions. In this context I address the perhaps most disputed and the most important question of the Slavic nominal inflectional morphology: whether there was in Proto-Slavic an Auslautgesetz (ALG), a law of final syllables, that narrowed the Proto-Indo-European vowel */o/ to */u/ in closed word-final syllables. In addition, the work contains an exhaustive morphological classification of the nouns and adjectives that occur in canonical Old Church Slavic. I argue that Proto-Indo-European */o/ became Proto-Slavic */u/ before word-final */s/ and */N/. This conclusion is based on the impossibility of finding credible analogical (as opposed to phonological) explanations for the forms supporting the ALG hypothesis, and on the survival of the neuter gender in Slavic. It is not likely that the */o/-stem nominative singular ending */-u/ was borrowed from the accusative singular, because the latter would have been the only paradigmatic form with the stem vowel */-u-/. It is equally unlikely that the ending */-u/ was borrowed from the */u/-stems, because the latter constituted a moribund class. The usually stated motivation for such an analogical borrowing, i.e. a need to prevent the merger of */o/-stem masculines with neuters of the same class, is not tenable. Extra-Slavic, as well as intra-Slavic evidence suggests that phonologically-triggered mergers between two semantically opaque genders do not tend to be prevented, but rather that such mergers lead to the loss of the gender opposition in question. On the other hand, if */-os/ had not become */-us/, most nouns and, most importantly, all adjectives and pronouns would have lost the formal distinction between masculines and neuters. This would have necessarily resulted in the loss of the neuter gender. A new explanation is given for the most apparent piece of evidence against the ALG hypothesis, the nominative-accusative singular of the */es/-stem neuters, e.g. nebo 'sky'. I argue that it arose in late Proto-Slavic dialects, replacing regular nebe, under the influence of the */o/- and */yo/-stems where a correlation had emerged between a hard root-final consonant and the termination -o, on the one hand, and a soft root-final consonant and the termination -e, on the other.

Using Mathematical Morphology for Geometric Music Retrieval

The usual task in music information retrieval (MIR) is to find occurrences of a monophonic query pattern within a music database, which can contain both monophonic and polyphonic content. The so-called query-by-humming systems are a famous instance of content-based MIR. In such a system, the user's hummed query is converted into symbolic form to perform search operations in a similarly encoded database. The symbolic representation (e.g., textual, MIDI or vector data) is typically a quantized and simplified version of the sampled audio data, yielding to faster search algorithms and space requirements that can be met in real-life situations. In this thesis, we investigate geometric approaches to MIR. We first study some musicological properties often needed in MIR algorithms, and then give a literature review on traditional (e.g., string-matching-based) MIR algorithms and novel techniques based on geometry. We also introduce some concepts from digital image processing, namely the mathematical morphology, which we will use to develop and implement four algorithms for geometric music retrieval. The symbolic representation in the case of our algorithms is a binary 2-D image. We use various morphological pre- and post-processing operations on the query and the database images to perform template matching / pattern recognition for the images. The algorithms are basically extensions to classic image correlation and hit-or-miss transformation techniques used widely in template matching applications. They aim to be a future extension to the retrieval engine of C-BRAHMS, which is a research project of the Department of Computer Science at University of Helsinki.

Missing-In-Metastasis (MIM) regulates cell morphology by promoting plasma membrane and actin cytoskeleton dynamics

The cells of multicellular organisms have differentiated to carry out specific functions that are often accompanied by distinct cell morphology. The actin cytoskeleton is one of the key regulators of cell shape subsequently controlling multiple cellular events including cell migration, cell division, endo- and exocytosis. A large set of actin regulating proteins has evolved to achieve and tightly coordinate this wide range of functions. Some actin regulator proteins have so-called house keeping roles and are essential for all eukaryotic cells, but some have evolved to meet the requirements of more specialized cell-types found in higher organisms enabling complex functions of differentiated organs, such as liver, kidney and brain. Often processes mediated by the actin cytoskeleton, like formation of cellular protrusions during cell migration, are intimately linked to plasma membrane remodeling. Thus, a close cooperation between these two cellular compartments is necessary, yet not much is known about the underlying molecular mechanisms. This study focused on a vertebrate-specific protein called missing-in-metastasis (MIM), which was originally characterized as a metastasis suppressor of bladder cancer. We demonstrated that MIM regulates the dynamics of actin cytoskeleton via its WH2 domain, and is expressed in a cell-type specific manner. Interestingly, further examination showed that the IM-domain of MIM displays a novel membrane tubulation activity, which induces formation of filopodia in cells. Following studies demonstrated that this membrane deformation activity is crucial for cell protrusions driven by MIM. In mammals, there are five members of IM-domain protein family. Functions and expression patterns of these family members have remained poorly characterized. To understand the physiological functions of MIM, we generated MIM knockout mice. MIM-deficient mice display no apparent developmental defects, but instead suffer from progressive renal disease and increased susceptibility to tumors. This indicates that MIM plays a role in the maintenance of specific physiological functions associated with distinct cell morphologies. Taken together, these studies implicate MIM both in the regulation of the actin cytoskeleton and the plasma membrane. Our results thus suggest that members of MIM/IRSp53 protein family coordinate the actin cytoskeleton:plasma membrane interface to control cell and tissue morphogenesis in multicellular organisms.

Exotic superfluid states of ultra-cold Fermi gases

This is a study of ultra-cold Fermi gases in different systems. This thesis is focused on exotic superfluid states, for an example on the three component Fermi gas and the FFLO phase in optical lattices. In the two-components case, superfluidity is studied mainly in the case of the spin population imbalanced Fermi gases and the phase diagrams are calculated from the mean-field theory. Different methods to detect different phases in optical lattices are suggested. In the three-component case, we studied also the uniform gas and harmonically trapped system. In this case, the BCS theory is generalized to three-component gases. It is also discussed how to achieve the conditions to get an SU(3)-symmetric Hamiltonian in optical lattices. The thesis is divided in chapters as follows: Chapter 1 is an introduction to the field of cold quantum gases. In chapter 2 optical lattices and their experimental characteristics are discussed. Chapter 3 deals with two-components Fermi gases in optical lattices and the paired states in lattices. In chapter 4 three-component Fermi gases with and without a harmonic trap are explored, and the pairing mechanisms are studied. In this chapter, we also discuss three-component Fermi gases in optical lattices. Chapter 5 devoted to the higher order correlations, and what they can tell about the paired states. Chapter 6 concludes the thesis.

Effects of climate and morphology on temperature conditions of lakes

To a large extent, lakes can be described with a one-dimensional approach, as their main features can be characterized by the vertical temperature profile of the water. The development of the profiles during the year follows the seasonal climate variations. Depending on conditions, lakes become stratified during the warm summer. After cooling, overturn occurs, water cools and an ice cover forms. Typically, water is inversely stratified under the ice, and another overturn occurs in spring after the ice has melted. Features of this circulation have been used in studies to distinguish between lakes in different areas, as basis for observation systems and even as climate indicators. Numerical models can be used to calculate temperature in the lake, on the basis of the meteorological input at the surface. The simple form is to solve the surface temperature. The depth of the lake affects heat transfer, together with other morphological features, the shape and size of the lake. Also the surrounding landscape affects the formation of the meteorological fields over the lake and the energy input. For small lakes the shading by the shores affects both over the lake and inside the water body bringing limitations for the one-dimensional approach. A two-layer model gives an approximation for the basic stratification in the lake. A turbulence model can simulate vertical temperature profile in a more detailed way. If the shape of the temperature profile is very abrupt, vertical transfer is hindered, having many important consequences for lake biology. One-dimensional modelling approach was successfully studied comparing a one-layer model, a two-layer model and a turbulence model. The turbulence model was applied to lakes with different sizes, shapes and locations. Lake models need data from the lakes for model adjustment. The use of the meteorological input data on different scales was analysed, ranging from momentary turbulent changes over the lake to the use of the synoptical data with three hour intervals. Data over about 100 past years were used on the mesoscale at the range of about 100 km and climate change scenarios for future changes. Increasing air temperature typically increases water temperature in epilimnion and decreases ice cover. Lake ice data were used for modelling different kinds of lakes. They were also analyzed statistically in global context. The results were also compared with results of a hydrological watershed model and data from very small lakes for seasonal development.

Characterisation of cellulose- and lignin-based materials using x-ray scattering methods

Wood is an important material for the construction and pulping industries. Using x-ray diffraction the microfibril angle of Sitka spruce wood was studied in the first part of this thesis. Sitka spruce (Picea sitchensis [Bong.] Carr.) is native to the west coast of North America, but due to its fast growth rate, it has also been imported to Europe. So far, its nanometre scale properties have not been systematically characterised. In this thesis the microfibril angle of Sitka spruce was shown to depend significantly on the origin of the tree in the first annual rings near the pith. Wood can be further processed to separate lignin from cellulose and hemicelluloses. Solid cellulose can act as a reducer for metal ions and it is also a porous support for nanoparticles. By chemically reducing nickel or copper in the solid cellulose support it is possible to get small nanoparticles on the surfaces of the cellulose fibres. Cellulose supported metal nanoparticles can potentially be used as environmentally friendly catalysts in organic chemistry reactions. In this thesis the size of the nickel and copper containing nanoparticles were studied using anomalous small-angle x-ray scattering and wide-angle x-ray scattering. The anomalous small-angle x-ray scattering experiments showed that the crystallite size of the copper oxide nanoparticles was the same as the size of the nanoparticles, so the nanoparticles were single crystals. The nickel containing nanoparticles were amorphous, but crystallised upon heating. The size of the nanoparticles was observed to be smaller when the reduction of nickel was done in aqueous ammonium hydrate medium compared to reduction made in aqueous solution. Lignin is typically seen as the side-product of wood industries. Lignin is the second most abundant natural polymer on Earth, and it possesses potential to be a useful material for many purposes in addition to being an energy source for the pulp mills. In this thesis, the morphology of several lignins, which were produced by different separation methods from wood, was studied using small-angle and ultra small-angle x-ray scattering. It was shown that the fractal model previously proposed for the lignin structure does not apply to most of the extracted lignin types. The only lignin to which the fractal model could be applied was kraft lignin. In aqueous solutions the average shape of the low molar mass kraft lignin particles was observed to be elongated and flat. The average shape does not necessarily correspond to the shape of the individual particles because of the polydispersity of the fraction and due to selfassociation of the particles. Lignins, and especially lignosulfonate, have many uses as dispersants, binders and emulsion stabilisers. In this thesis work the selfassociation of low molar mass lignosulfonate macromolecules was observed using small-angle x-ray scattering. By taking into account the polydispersity of the studied lignosulfonate fraction, the shape of the lignosulfonate particles was determined to be flat by fitting an oblate ellipsoidal model to the scattering intensity.

Toward a widely usable finite-state morphology workbench for less studied languages, 1: Desiderata

Most of the world’s languages lack electronic word form dictionaries. The linguists who gather such dictionaries could be helped with an efficient morphology workbench that adapts to different environments and uses. A widely usable workbench could be characterized, ideally, as generally applicable, extensible, and freely available (GEA). It seems that such a solution could be implemented in the framework of finite-state methods. The current work defines the GEA desiderata and starts a series of articles concerning these desiderata in finite- state morphology. Subsequent parts will review the state of the art and present an action plan toward creating a widely usable finite-state morphology workbench.

Root system traits of Norway spruce, Scots pine, and silver birch in mixed boreal forests: an analysis of root architecture, morphology, and anatomy

The aim of this thesis was to unravel the functional-structural characteristics of root systems of Betula pendula Roth., Picea abies (L.) Karst., and Pinus sylvestris L. in mixed boreal forest stands differing in their developmental stage and site fertility. The root systems of these species had similar structural regularities: horizontally-oriented shallow roots defined the horizontal area of influence, and within this area, each species placed fine roots in the uppermost soil layers, while sinker roots defined the maximum rooting depth. Large radial spread and high ramification of coarse roots, and the high specific root length (SRL) and root length density (RLD) of fine roots indicated the high belowground competitiveness and root plasticity of B. pendula. Smaller radial root spread and sparser branching of coarse roots, and low SRL and RLD of fine roots of the conifers could indicate their more conservative resource use and high association with and dependence on ectomycorrhiza-forming fungi. The vertical fine root distributions of the species were mostly overlapping, implying the possibility for intense belowground competition for nutrients. In each species, conduits tapered and their frequency increased from distal roots to the stem, from the stem to the branches, and to leaf petioles in B. pendula. Conduit tapering was organ-specific in each species violating the assumptions of the general vascular scaling model (WBE). This reflects the hierarchical organization of a tree and differences between organs in the relative importance of transport, safety, and mechanical demands. The applied root model was capable of depicting the mass, length and spread of coarse roots of B. pendula and P. abies, and to the lesser extent in P. sylvestris. The roots did not follow self-similar fractal branching, because the parameter values varied within the root systems. Model parameters indicate differences in rooting behavior, and therefore different ecophysiological adaptations between species.