Ultrasound-assisted surface engineering of pharmaceutical powders

Effective processing of powdered particles can facilitate powder handling and result in better drug product performance, which is of great importance in the pharmaceutical industry where the majority of active pharmaceutical ingredients (APIs) are delivered as solid dosage forms. The purpose of this work was to develop a new ultrasound-assisted method for particle surface modification and thin-coating of pharmaceutical powders. The ultrasound was used to produce an aqueous mist with or without a coating agent. By using the proposed technique, it was possible to decrease the interparticular interactions and improve rheological properties of poorly-flowing water-soluble powders by aqueous smoothing of the rough surfaces of irregular particles. In turn, hydrophilic polymer thin-coating of a hydrophobic substance diminished the triboelectrostatic charge transfer and improved the flowability of highly cohesive powder. To determine the coating efficiency of the technique, the bioactive molecule β-galactosidase was layered onto the surface of powdered lactose particles. Enzyme-treated materials were analysed by assaying the quantity of the reaction product generated during enzymatic cleavage of the milk sugar. A near-linear increase in the thickness of the drug layer was obtained during progressive treatment. Using the enzyme coating procedure, it was confirmed that the ultrasound-assisted technique is suitable for processing labile protein materials. In addition, this pre-treatment of milk sugar could be used to improve utilization of lactose-containing formulations for populations suffering from severe lactose intolerance. Furthermore, the applicability of the thin-coating technique for improving homogeneity of low-dose solid dosage forms was shown. The carrier particles coated with API gave rise to uniform distribution of the drug within the powder. The mixture remained homogeneous during further tabletting, whereas the reference physical powder mixture was subject to segregation. In conclusion, ultrasound-assisted surface engineering of pharmaceutical powders can be effective technology for improving formulation and performance of solid dosage forms such as dry powder inhalers (DPI) and direct compression products.

Lignocellulose degradation and humus modification by the fungus Paecilomyces inflatus

Composting is the biological conversion of solid organic waste into usable end products such as fertilizers, substrates for mushroom production and biogas. Although composts are highly variable in their bulk composition, composting material is generally based on lignocellulose compounds derived from agricultural, forestry, fruit and vegetable processing, household and municipal wastes. Lignocellulose is very recalcitrant; however it is rich and abundant source of carbon and energy. Therefore lignocellulose degradation is essential for maintaining the global carbon cycle. In compost, the active component involved in the biodegradation and conversion processes is the resident microbial population, among which microfungi play a very important role. In composting pile the warm, humid, and aerobic environment provides the optimal conditions for their development. Microfungi use many carbon sources, including lignocellulosic polymers and can survive in extreme conditions. Typically microfungi are responsible for compost maturation. In order to improve the composting process, more information is needed about the microbial degradation process. Better knowledge on the lignocellulose degradation by microfungi could be used to optimize the composting process. Thus, this thesis focused on lignocellulose and humic compounds degradation by a microfungus Paecilomyces inflatus, which belongs to a flora of common microbial compost, soil and decaying plant remains. It is a very common species in Europe, North America and Asia. The lignocellulose and humic compounds degradation was studied using several methods including measurements of carbon release from 14C-labelled compounds, such as synthetic lignin (dehydrogenative polymer, DHP) and humic acids, as well as by determination of fibre composition using chemical detergents and sulphuric acid. Spectrophotometric enzyme assays were conducted to detect extracellular lignocellulose-degrading hydrolytic and oxidative enzymes. Paecilomyces inflatus secreted clearly extracellular laccase to the culture media. Laccase was involved in the degradation process of lignin and humic acids. In compost P. inflatus mineralised 6-10% of 14C-labelled DHP into carbon dioxide. About 15% of labelled DHP was converted into water-soluble compounds. Also humic acids were partly mineralised and converted into water-soluble material, such as low-molecular mass fulvic acid-like compounds. Although laccase activity in aromatics-rich compost media clearly is connected with the degradation process of lignin and lignin-like compounds, it may preferentially effect the polymerisation and/or detoxification of such aromatic compounds. P. inflatus can degrade lignin and carbohydrates also while growing in straw and in wood. The cellulolytic enzyme system includes endoglucanase and β-glucosidase. In P. inflatus the secretion of these enzymes was stimulated by low-molecular-weight aromatics, such as soil humic acid and veratric acid. When strains of P. inflatus from different ecophysiological origins were compared, indications were found that specific adaptation strategies needed for lignocellulosics degradation may operate in P. inflatus. The degradative features of these microfungi are on relevance for lignocellulose decomposition in nature, especially in soil and compost environments, where basidiomycetes are not established. The results of this study may help to understand, control and better design the process of plant polymer conversion in compost environment, with a special emphasis on the role of ubiquitous microfungi.

Heparin-binding growth-associated molecule (HB-GAM) in activity-dependent neuronal plasticity in hippocampus

Cell adhesion and extracellular matrix (ECM) molecules play a significant role in neuronal plasticity both during development and in the adult. Plastic changes in which ECM components are implicated may underlie important nervous system functions, such as memory formation and learning. Heparin-binding growthassociated molecule (HB-GAM, also known as pleiotrophin), is an ECM protein involved in neurite outgrowth, axonal guidance and synaptogenesis during perinatal period. In the adult brain HB-GAM expression is restricted to the regions which display pronounced synaptic plasticity (e.g., hippocampal CA3-CA1 areas, cerebral cortex laminae II-IV, olfactory bulb). Expression of HB-GAM is regulated in an activity-dependent manner and is also induced in response to neuronal injury. In this work mutant mice were used to study the in vivo function of HB-GAM and its receptor syndecan-3 in hippocampal synaptic plasticity and in hippocampus-dependent behavioral tasks. Phenotypic analysis of HBGAM null mutants and mice overexpressing HB-GAM revealed that opposite genetic manipulations result in reverse changes in synaptic plasticity as well as behavior in the mutants. Electrophysiological recordings showed that mice lacking HB-GAM have an increased level of long-term potentiation (LTP) in the area CA1 of hippocampus and impaired spatial learning, whereas animals with enhanced level of HB-GAM expression have attenuated LTP, but outperformed their wild-type controls in spatial learning. It was also found that GABA(A) receptor-mediated synaptic transmission is altered in the transgenic mice overexpressing HB-GAM. The results suggest that these animals have accentuated hippocampal GABAergic inhibition, which may contribute to the altered glutamatergic synaptic plasticity. Structural studies of HB-GAM demonstrated that this protein belongs to the thrombospondin type I repeat (TSR) superfamily and contains two β-sheet domains connected by a flexible linker. It was found that didomain structure is necessary for biological activity of HB-GAM and electrophysiological phenotype displayed by the HB-GAM mutants. The individual domains displayed weaker binding to heparan sulfate and failed to promote neurite outgrowth as well as affect hippocampal LTP. Effects of HB-GAM on hippocampal synaptic plasticity are believed to be mediated by one of its (co-)receptor molecules, namely syndecan-3. In support of that, HB-GAM did not attenuate LTP in mice deficient in syndecan-3 as it did in wild-type controls. In addition, syndecan-3 knockout mice displayed electrophysiological and behavioral phenotype similar to that of HB-GAM knockouts (i.e. enhanced LTP and impaired learning in Morris water-maze). Thus HB-GAM and syndecan-3 are important modulators of synaptic plasticity in hippocampus and play a role in regulation of learning-related behavior.

Synthesis of neoglycoconjugates and oligosaccharides with potential anti-Helicobacter pylori activity

The significance of carbohydrate-protein interactions in many biological phenomena is now widely acknowledged and carbohydrate based pharmaceuticals are under intensive development. The interactions between monomeric carbohydrate ligands and their receptors are usually of low affinity. To overcome this limitation natural carbohydrate ligands are often organized as multivalent structures. Therefore, artificial carbohydrate pharmaceuticals should be constructed on the same concept, as multivalent carbohydrates or glycoclusters. Infections of specific host tissues by bacteria, viruses, and fungi are among the unfavorable disease processes for which suitably designed carbohydrate inhibitors represent worthy targets. The bacterium Helicobacter pylori colonizes more than half of all people worldwide, causing gastritis, gastric ulcer, and conferring a greater risk of stomach cancer. The present medication therapy for H. pylori includes the use of antibiotics, which is associated with increasing incidence of bacterial resistance to traditional antibiotics. Therefore, the need for an alternative treatment method is urgent. In this study, four novel synthesis procedures of multivalent glycoconjugates were created. Three different scaffolds representing linear (chondroitin oligomer), cyclic (γ-cyclodextrin), and globular (dendrimer) molecules were used. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc all representing analogues of the tissue binding epitopes for H. pylori. The first synthetic method included the reductive amination of scaffold molecules modified to express primary amine groups, and in the case of dendrimer direct amination to scaffold molecule presenting 64 primary amine groups. The second method described a direct procedure for amidation of glycosylamine modified oligosaccharides to scaffold molecules presenting carboxyl groups. The final two methods that were created both included an oxime-linkage on linkers of different length. All the new synthetic procedures synthesized had the advantage of using unmodified reducing sugars as starting material making it easy to synthesize glycoconjugates of different specificity. In addition, the binding activity of an array of neoglycolipids to H. pylori was studied. Consequently, two new neolacto-based structures, Glcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer and GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer, with binding activity toward H. pylori were discovered. Interestingly, N-methyl and N-ethyl amide modification of the GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer glucuronic acid residue resulted in more effective H. pylori binding epitopes than the parent molecule.

Functional and cellular analysis of autoimmune regulator (AIRE) protein

Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.

The effect of lignin content and lignin modification on Norway spruce wood properties and decay resistance

Three different Norway spruce cutting clones growing in three environments with different soil and climatic conditions were studied. The purpose was to follow variation in the radial growth rate, wood properties and lignin content and to modify wood lignin with a natural monolignol, coniferyl alcohol, by making use of inherent wood peroxidases. In addition, the incorporation of chlorinated anilines into lignin was studied with synthetic model compounds and synthetic lignin preparations to show whether unnatural compounds originating from pesticides could be bound in the lignin polymer. The lignin content of heartwood, sapwood and earlywood was determined by applying Fourier transform infrared (FTIR) spectroscopy and a principal component regression (PCR) technique. Wood blocks were treated with coniferyl alcohol by using a vacuum impregnation method. The effect of impregnation was assessed by FTIR and by a fungal decay test. Trees from a fertile site showed the highest growth rate and sapwood lignin content and the lowest latewood proportion, weight density and modulus of rupture (MOR). Trees from a medium fertile site had the lowest growth rate and the highest latewood proportion, weight density, modulus of elasticity (MOE) and MOR. The most rapidly growing clone showed the lowest latewood proportion, weight density, MOE and MOR. The slowest growing clone had the lowest sapwood lignin content and the highest latewood proportion, weight density, MOE and MOR. Differences between the sites and clones were small, while fairly large variation was found between the individual trees and growing seasons. The cutting clones maintained clone-dependent wood properties in the different growing sites although variation between trees was high and climatic factors affected growth. The coniferyl alcohol impregnation increased the content of different lignin-type phenolic compounds in the wood as well as wood decay resistance against a white-rot fungus, Coriolus versicolor. During the synthetic lignin preparation 3,4-dichloroaniline became bound by a benzylamine bond to β-O-4 structures in the polymer and it could not be released by mild acid hydrolysis. The natural monolignol, coniferyl alcohol, and chlorinated anilines could be incorporated into the lignin polymer in vivo and in vitro, respectively.

Growth and Modification of Cluster-Assembled Thin Films

Thin film applications have become increasingly important in our search for multifunctional and economically viable technological solutions of the future. Thin film coatings can be used for a multitude of purposes, ranging from a basic enhancement of aesthetic attributes to the addition of a complex surface functionality. Anything from electronic or optical properties, to an increased catalytic or biological activity, can be added or enhanced by the deposition of a thin film, with a thickness of only a few atomic layers at the best, on an already existing surface. Thin films offer both a means of saving in materials and the possibility for improving properties without a critical enlargement of devices. Nanocluster deposition is a promising new method for the growth of structured thin films. Nanoclusters are small aggregates of atoms or molecules, ranging in sizes from only a few nanometers up to several hundreds of nanometers in diameter. Due to their large surface to volume ratio, and the confinement of atoms and electrons in all three dimensions, nanoclusters exhibit a wide variety of exotic properties that differ notably from those of both single atoms and bulk materials. Nanoclusters are a completely new type of building block for thin film deposition. As preformed entities, clusters provide a new means of tailoring the properties of thin films before their growth, simply by changing the size or composition of the clusters that are to be deposited. Contrary to contemporary methods of thin film growth, which mainly rely on the deposition of single atoms, cluster deposition also allows for a more precise assembly of thin films, as the configuration of single atoms with respect to each other is already predetermined in clusters. Nanocluster deposition offers a possibility for the coating of virtually any material with a nanostructured thin film, and therein the enhancement of already existing physical or chemical properties, or the addition of some exciting new feature. A clearer understanding of cluster-surface interactions, and the growth of thin films by cluster deposition, must, however, be achieved, if clusters are to be successfully used in thin film technologies. Using a combination of experimental techniques and molecular dynamics simulations, both the deposition of nanoclusters, and the growth and modification of cluster-assembled thin films, are studied in this thesis. Emphasis is laid on an understanding of the interaction between metal clusters and surfaces, and therein the behaviour of these clusters during deposition and thin film growth. The behaviour of single metal clusters, as they impact on clean metal surfaces, is analysed in detail, from which it is shown that there exists a cluster size and deposition energy dependent limit, below which epitaxial alignment occurs. If larger clusters are deposited at low energies, or cluster-surface interactions are weaker, non-epitaxial deposition will take place, resulting in the formation of nanocrystalline structures. The effect of cluster size and deposition energy on the morphology of cluster-assembled thin films is also determined, from which it is shown that nanocrystalline cluster-assembled films will be porous. Modification of these thin films, with the purpose of enhancing their mechanical properties and durability, without destroying their nanostructure, is presented. Irradiation with heavy ions is introduced as a feasible method for increasing the density, and therein the mechanical stability, of cluster-assembled thin films, without critically destroying their nanocrystalline properties. The results of this thesis demonstrate that nanocluster deposition is a suitable technique for the growth of nanostructured thin films. The interactions between nanoclusters and their supporting surfaces must, however, be carefully considered, if a controlled growth of cluster-assembled thin films, with precisely tailored properties, is to be achieved.

Optical Tweezers for Single Molecule Biology

Molecular machinery on the micro-scale, believed to be the fundamental building blocks of life, involve forces of 1-100 pN and movements of nanometers to micrometers. Micromechanical single-molecule experiments seek to understand the physics of nucleic acids, molecular motors, and other biological systems through direct measurement of forces and displacements. Optical tweezers are a popular choice among several complementary techniques for sensitive force-spectroscopy in the field of single molecule biology. The main objective of this thesis was to design and construct an optical tweezers instrument capable of investigating the physics of molecular motors and mechanisms of protein/nucleic-acid interactions on the single-molecule level. A double-trap optical tweezers instrument incorporating acousto-optic trap-steering, two independent detection channels, and a real-time digital controller was built. A numerical simulation and a theoretical study was performed to assess the signal-to-noise ratio in a constant-force molecular motor stepping experiment. Real-time feedback control of optical tweezers was explored in three studies. Position-clamping was implemented and compared to theoretical models using both proportional and predictive control. A force-clamp was implemented and tested with a DNA-tether in presence of the enzyme lambda exonuclease. The results of the study indicate that the presented models describing signal-to-noise ratio in constant-force experiments and feedback control experiments in optical tweezers agree well with experimental data. The effective trap stiffness can be increased by an order of magnitude using the presented position-clamping method. The force-clamp can be used for constant-force experiments, and the results from a proof-of-principle experiment, in which the enzyme lambda exonuclease converts double-stranded DNA to single-stranded DNA, agree with previous research. The main objective of the thesis was thus achieved. The developed instrument and presented results on feedback control serve as a stepping stone for future contributions to the growing field of single molecule biology.

The neuronal cell adhesion molecule ICAM-5

The neuronal cell adhesion molecule ICAM-5 ICAM-5 (telencephalin) belongs to the intercellular adhesion molecule (ICAM)-subgroup of the immunoglobulin superfamily (IgSF). ICAMs participate in leukocyte adhesion and adhesion-dependent functions in the central nervous system (CNS) through interacting with the leukocyte-specific b2 integrins. ICAM-5 is found in the mammalian forebrain, appears at the time of birth, and is located at the cell soma and neuronal dendrites. Recent studies also show that it is important for the regulation of immune functions in the brain and for the development and maturation of neuronal synapses. The clinical importance of ICAM-5 is still under investigation; it may have a role in the development of Alzheimer s disease (AD). In this study, the role of ICAM-5 in neuronal differentiation and its associations with a-actinin and N-methyl-D-aspartic acid (NMDA) receptors were examined. NMDA receptors (NMDARs) are known to be involved in many neuronal functions, including the passage of information from one neuron to another one, and thus it was thought important to study their role related to ICAM-5. The results suggested that ICAM-5 was able to induce dendritic outgrowth through homophilic adhesion (ICAM-5 monomer binds to another ICAM-5 monomer in the same or neighbouring cell), and the homophilic binding activity appeared to be regulated by monomer/multimer transition. Moreover, ICAM-5 binding to a-actinin was shown to be important for neuritic outgrowth. It was examined whether matrix metalloproteinases (MMPs) are the main enzymes involved in ICAM-5 ectodomain cleavage. The results showed that stimulation of NMDARs leads to MMP activation, cleavage of ICAM-5 and it is accompanied by dendritic spine maturation. These findings also indicated that ICAM-5 and NMDA receptor subunit 1 (NR1) compete for binding to a-actinin, and ICAM-5 may regulate the NR1 association with the actin cytoskeleton. Thus, it is concluded that ICAM-5 is a crucial cell adhesion molecule involved in the development of neuronal synapses, especially in the regulation of dendritic spine development, and its functions may also be involved with memory formation and learning.