Distribution and adhesion-promoting activity of alpha4 and alpha5 chain laminins in human endothelia and carcinomas

Basement membranes are specialized sheets of extracellular matrix found in contact with epithelia, endothelia, and certain isolated cells. They support tissue architecture and regulate cell behaviour. Laminins are among the main constituents of basement membranes. Due to differences between laminin isoforms, laminins confer structural and functional diversity to basement membranes. The first aim of this study was to gain insights into the potential functions of the then least characterized laminins, alpha4 chain laminins, by evaluating their distribution in human tissues. We thus created a monoclonal antibody specific for laminin alpha4 chain. By immunohistochemistry, alpha4 chain laminins were primarily localized to basement membranes of blood vessel endothelia, skeletal, heart, and smooth muscle cells, nerves, and adipocytes. In addition, alpha4 chain laminins were found in the region of certain epithelial basement membranes in the epidermis, salivary gland, pancreas, esophagus, stomach, intestine, and kidney. Because of the consistent presence of alpha4 chain laminins in endothelial basement membranes of blood vessels, we evaluated the potential roles of endothelial laminins in blood vessels, lymphatic vessels, and carcinomas. Human endothelial cells produced alpha4 and alpha5 chain laminins. In quantitative and morphological adhesion assays, human endothelial cells barely adhered to alpha4 chain-containing laminin-411. The weak interaction of endothelial cells with laminin-411 appeared to be mediated by alpha6beta1 integrin. The alpha5 chain-containing laminin-511 promoted endothelial cell adhesion better than laminin-411, but it did not promote the formation of cell-extracellular matrix adhesion complexes. The adhesion of endothelial cells to laminin-511 appeared to be mediated by Lutheran glycoprotein together with beta1 and alphavbeta3 integrins. The results suggest that these laminins may induce a migratory phenotype in endothelial cells. In lymphatic capillaries, endothelial basement membranes showed immunoreactivity for laminin alpha4, beta1, beta2, and gamma1 chains, type IV and XVIII collagens, and nidogen-1. Considering the assumed inability of alpha4 chain laminins to polymerize and to promote basement membrane assembly, the findings may in part explain the incomplete basement membrane formation in these vessels. Lymphatic capillaries of ovarian carcinomas showed immunoreactivity also for laminin alpha5 chain and its receptor Lutheran glycoprotein, emphasizing a difference between normal and ovarian carcinoma lymphatic capillaries. In renal cell carcinomas, immunoreactivity for laminin alpha4 chain was found in stroma and basement membranes of blood vessels. In most tumours, immunoreactivity for laminin alpha4 chain was also observed in the basement membrane region of tumour cell islets. Renal carcinoma cells produced alpha4 chain laminins. Laminin-411 did not promote adhesion of renal carcinoma cells, but inhibited their adhesion to fibronectin. Renal carcinoma cells migrated more on laminin-411 than on fibronectin. The results suggest that alpha4 chain laminins have a counteradhesive function, and may thus have a role in detachment and invasion of renal carcinoma cells.

Role of Basement Membrane and Extracellular Matrix Proteins in the Adhesion and Spreading of Immortalized Human Corneal Epithelial Cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. Basement membrane (BM) and extracellular matrix (ECM) proteins function in these processes via integrin and non-integrin receptors. We have studied the adhesion, spreading and migration of immortalized human corneal epithelial (HCE) cells and their interactions with the laminins (Lms), fibronectins and tenascins produced. Human corneal BM expresses Lms-332 and -511, while Lm-111 was not found in these experiments. HCE cells produced both processed and unprocessed Lm-332, whereas neither Lm-111 nor Lm-511 was produced. Because HCE cells did not produce Lm-511, although it was present in corneal BM, we suggest that Lm-511 is produced by stromal keratocytes. The adhesion of HCE cells to Lms-111, -332 and -511 was studied first by determining the receptor composition of HCE cells and then by using quantitative cell adhesion assays. Immunofluorescence studies revealed the presence of integrin α2, α3, α6, β1 and β4 subunits. Among the non-integrin receptors, Lutheran (Lu) was found on adhering HCE cells. The cells adhered via integrin α3β1 to both purified human Lms-332 and -511 as well as to endogenous Lm-332. However, only integrin β1 subunit functioned in HCE cell adhesion to mouse Lm-111. The adhesion of HCE cells to Lm-511 was also mediated by Lu. Since Lm-511 did not induce Lu into focal adhesions in HCE cells, we suggest that Lm-511 serves as an ECM ligand enabling cell motility. HCE cells produced extradomain-A fibronectin, oncofetal fibronectin and tenascin-C (Tn-C), which are also found during corneal wound healing. Monoclonal antibodies (MAbs) against integrins α5β1 and αvβ6 as well as the arginine-glycine-aspartic acid (RGD) peptide inhibited the adhesion of HCE cells to fibronectin. Although the cells did not adhere to Tn-C, they adhered to the fibronectin/Tn-C coat and were then more efficiently inhibited by the function-blocking MAbs and RGD peptide. During the early adhesion, HCE cells codeposited Lm-332 and the large subunit of tenascin-C (Tn-CL) beneath the cells via the Golgi apparatus and microtubules. Integrin β4 subunit, which is a hemidesmosomal component, did not mediate the early adhesion of HCE cells to Lm-332 or Lm-332/Tn-C. Based on these results, we suggest that the adhesion of HCE cells is initiated by Lm-332 and modulated by Tn-CL, as it has been reported to prevent the assembly of hemidesmosomes. Thereby, Tn-CL functions in the motility of HCE cells during wound healing. The different distribution of processed and unprocessed Lm-332 in adhering, spreading and migrating HCE cells suggests a distinct role for these isoforms. We conclude that the processed Lm-332 functions in cell adhesion, whereas the unprocessed Lm-332 participates in cell spreading and migration.

Adhesion,Presence and Antifouling of Deinococcus geothermalis in Paper Machine Environment

This thesis has two items: biofouling and antifouling in paper industry. Biofouling means unwanted microbial accumulation on surfaces causing e.g. disturbances in industrial processes, contamination of medical devices or of water distribution networks. Antifouling focuses on preventing accumulation of the biofilms in undesired places. Deinococcus geothermalis is a pink-pigmented, thermophilic bacterium, and extremely resistant towards radiation, UV-light and desiccation and known as a biofouler of paper machines forming firm and biocide resistant biofilms on the stainless steel surfaces. The compact structure of biofilm microcolonies of D. geothermalis E50051 and the adhesion into abiotic surfaces were investigated by confocal laser scanning microscope combined with carbohydrate specific fluorescently labelled lectins. The extracellular polymeric substance in D. geothermalis microcolonies was found to be a composite of at least five different glycoconjugates contributing to adhesion, functioning as structural elements, putative storages for water, gliding motility and likely also to protection. The adhesion threads that D. geothermalis seems to use to adhere on an abiotic surface and to anchor itself to the neighbouring cells were shown to be protein. Four protein components of type IV pilin were identified. In addition, the lectin staining showed that the adhesion threads were covered with galactose containing glycoconjugates. The threads were not exposed on planktic cells indicating their primary role in adhesion and in biofilm formation. I investigated by quantitative real-time PCR the presence of D. geothermalis in biofilms, deposits, process waters and paper end products from 24 paper and board mills. The primers designed for doing this were targeted to the 16S rRNA gene of D. geothermalis. We found D. geothermalis DNA from 9 machines, in total 16 samples of the 120 mill samples searched for. The total bacterial content varied in those samples between 107 to 3 ×1010 16S rRNA gene copies g-1. The proportion of D. geothermalis in those same samples was minor, 0.03 1.3 % of the total bacterial content. Nevertheless D. geothermalis may endanger paper quality as its DNA was shown in an end product. As an antifouling method towards biofilms we studied the electrochemical polarization. Two novel instruments were designed for this work. The double biofilm analyzer was designed for search for a polarization program that would eradicate D. geothermalis biofilm or from stainless steel under conditions simulating paper mill environment. The Radbox instrument was designed to study the generation of reactive oxygen species during the polarization that was effective in antifouling of D. geothermalis. We found that cathodic character and a pulsed mode of polarization were required to achieve detaching D. geothermalis biofilm from stainless steel. We also found that the efficiency of polarization was good on submerged, and poor on splash area biofilms. By adding oxidative biocides, bromochloro-5,5-dimethylhydantoin, 2,2-dibromo-2-cyanodiacetamide or peracetic acid gave additive value with polarization, being active on splash area biofilms. We showed that the cathodically weighted pulsed polarization that was active in removing D. geothermalis was also effective in generation of reactive oxygen species. It is possible that the antifouling effect relied on the generation of ROS on the polarized steel surfaces. Antifouling method successful towards D. geothermalis that is a tenacious biofouler and possesses a high tolerance to oxidative stressors could be functional also towards other biofoulers and applicable in wet industrial processes elsewhere.

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.

Interfacial Phenomena in Pharmaceutical Low Moisture Content Powder Processing

Powders are essential materials in the pharmaceutical industry, being involved in majority of all drug manufacturing. Powder flow and particle size are central particle properties addressed by means of particle engineering. The aim of the thesis was to gain knowledge on powder processing with restricted liquid addition, with a primary focus on particle coating and early granule growth. Furthermore, characterisation of this kind of processes was performed. A thin coating layer of hydroxypropyl methylcellulose was applied on individual particles of ibuprofen in a fluidised bed top-spray process. The polymeric coating improved the flow properties of the powder. The improvement was strongly related to relative humidity, which can be seen as an indicator of a change in surface hydrophilicity caused by the coating. The ibuprofen used in the present study had a d50 of 40 μm and thus belongs to the Geldart group C powders, which can be considered as challenging materials in top-spray coating processes. Ibuprofen was similarly coated using a novel ultrasound-assisted coating method. The results were in line with those obtained from powders coated in the fluidised bed process mentioned above. It was found that the ultrasound-assisted method was capable of coating single particles with a simple and robust setup. Granule growth in a fluidised bed process was inhibited by feeding the liquid in pulses. The results showed that the length of the pulsing cycles is of importance, and can be used to adjust granule growth. Moreover, pulsed liquid feed was found to be of greater significance to granule growth in high inlet air relative humidity. Liquid feed pulsing can thus be used as a tool in particle size targeting in fluidised bed processes and in compensating for changes in relative humidity of the inlet air. The nozzle function of a two-fluid external mixing pneumatic nozzle, typical for small scale pharmaceutical fluidised bed processes, was studied in situ in an ongoing fluidised bed process with particle tracking velocimetry. It was found that the liquid droplets undergo coalescence as they proceed away from the nozzle head. The coalescence was expected to increase droplet speed, which was confirmed in the study. The spray turbulence was studied, and the results showed turbulence caused by the event of atomisation and by the oppositely directed fluidising air. It was concluded that particle tracking velocimetry is a suitable tool for in situ spray characterisation. The light transmission through dense particulate systems was found to carry information on particle size and packing density as expected based on the theory of light scattering by solids. It was possible to differentiate binary blends consisting of components with differences in optical properties. Light transmission showed potential as a rapid, simple and inexpensive tool in characterisation of particulate systems giving information on changes in particle systems, which could be utilised in basic process diagnostics.

Tuning the interfacial properties of supported metal nanoclusters

Nanoclusters are objects made up of several to thousands of atoms and form a transitional state of matter between single atoms and bulk materials. Due to their large surface-to-volume ratio, nanoclusters exhibit exciting and yet poorly studied size dependent properties. When deposited directly on bare metal surfaces, the interaction of the cluster with the substrate leads to alteration of the cluster properties, making it less or even non-functional. Surfaces modified with self-assembled monolayers (SAMs) were shown to form an interesting alternative platform, because of the possibility to control wettability by decreasing the surface reactivity and to add functionalities to pre-formed nanoclusters. In this thesis, the underlying size effects and the influence of the nanocluster environment are investigated. The emphasis is on the structural and magnetic properties of nanoclusters and their interaction with thiol SAMs. We report, for the first time, a ferromagnetic-like spin-glass behaviour of uncapped nanosized Au islands tens of nanometres in size. The flattening kinetics of the nanocluster deposition on thiol SAMs are shown to be mediated mainly by the thiol terminal group, as well as the deposition energy and the particle size distribution. On the other hand, a new mechanism for the penetration of the deposited nanoclusters through the monolayers is presented, which is fundamentally different from those reported for atom deposition on alkanethiols. The impinging cluster is shown to compress the thiol layer against the Au surface and subsequently intercalate at the thiol-Au interface. The compressed thiols try then to straighten and push the cluster away from the surface. Depending on the cluster size, this restoring force may or may not enable a covalent cluster-surface bond formation, giving rise to various cluster-surface binding patterns. Compression and straightening of the thiol molecules pinpoint the elastic nature of the SAMs, which has been investigated in this thesis using nanoindentation. The nanoindenation method has been applied to SAMs of varied tail groups, giving insight into the mechanical properties of thiol modified metal surfaces.