The airway epithelium plays a crucial role in protecting the underlying connective tissue (CT) from noxious agents. Damage and shedding of the epithelium are observed in the airways of asthma, cystic fibrosis and rhinitis patients. The aim of this study was to investigate the mechanisms by which epithelial damage occurs, and the consequences of such damage for the inflammatory process in the airways. In this study, cultured normal human bronchial epithelial cells, excised rat tracheae, and cultured murine mast cells were used as model systems. Metabolic alterations, morphological changes and cell-cell contact stabilities were investigated. The T-helper (Th)-1 cytokines, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin (IL)-1β were found to be pro-inflammatory, leading to major morphological changes, inhibitions in desmosome formation, and accelerated cell death. The Th2 cytokines, IL-4, IL-5, and IL-13 were found to cause no changes in cell death, nitric oxide levels and desmosome formation but instead an increase in proliferation, therefore were anti-inflammatory in this respect. Increasing the osmolarity of the airway surface liquid (ASL) altered the integrity of the tight junction (TJ) and allowed a 4-kDa compound to penetrate the epithelial layer and access the CT. This effect was reversible if the ASL was returned to 295 mOsm. Intentionally breaking the TJ with EGTA and subsequent osmolar changes in ASL demonstrated the importance of TJ and the fragility of the CT under hyperosmolar stress, leading to a disrupted CT with larger capillaries and altered elemental ion content and epithelial denudation. Hydrocortisone was shown to downregulate IL-4-induced IL-6 upregulation in murine mast cells. Furthermore, incubating mast cells with hydrocortisone lead to a new subpopulation that was morphologically unique, that displayed new cell surface markers (CD44 and CD61) and that lacked CD54. These changes modify the interactions of mast cells with surrounding cells in the CT and epithelium. In conclusion, the balance between pro- and anti-inflammatory cytokines and ASL osmolarity may influence the role of the airway epithelium as a barrier. The pharmacological use of hyperosmolarity to disrupt TJ reversibly may help facilitate the delivery of drugs through the airway epithelium.

Studies of intestinal drug permeability have traditionally been performed in the colon-derived Caco-2 cell model. However, the permeability of these cell monolayers resembles that of the colon rather than that of the small intestine, which is the major site of drug absorption following oral administration. One aim of this thesis was therefore to develop a new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are conditionally immortalized with a temperature sensitive mutant of SV40T. These cells proliferate and form multilayers at 33°C. At cultivation temperatures of 37 – 39°C, they stop proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed functional tight junctions. The barrier properties of the tight junctions such as transepithelial electrical resistance and permeability to hydrophilic markers resembled those of the human small intestine in vivo. These cells lacked functional expression of drug transport proteins and can therefore be used as a model to study passive drug permeability unbiased by active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of the human jejunum for both incompletely and completely absorbed drugs, and the prediction of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables better predictions of drug permeability in the small intestine than does Caco-2. The studies of the paracellular route and its importance for intestinal drug permeability was also in focus in the second part of this thesis, in which a new principle for tight junction modulation was developed, based on the primary structure of the extracellular tight junction protein occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased the permeability of the tight junctions, but lacked apical effect. This problem was solved by conjugation of one peptide to a lipoamino acid, resulting in two diastereomers with different effects. The L-isomer had a sustained apical effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides constitute a new class of tight junction modulators that can enhance the tight junction permeability.

The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion. In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes. We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2s, H-2q and H-2f) mouse strains, silver induced only ANolA responses in H-2s and H-2q mice, but not in H-2f mice. Further studies showed that the resistance to silver-induced ANolA production in H-2f mice was inherited as a dominant trait. We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice. Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.

Celiaki, eller glutenintolerans, är en av våra vanligaste kroniska sjukdomar i barnaåren. Sjukdomen orsakar en kraftig inflammation i tunntarmens slemhinna efter intag av glutenhaltig föda hos personer med ärftlig benägenhet att utveckla celiaki. En frisk tarm är kraftigt veckad för att öka ytan för upptag av näringsämnen. Ytan består dessutom av åtskilliga fingerliknande utskott, s.k. villi, och mellan villi finns kryptorna där celldelning och celldifferentiering sker. Villi och kryptor kantas av epitelceller, enterocyter, vilkas uppgift är att ta upp näring från tarminnehållet samt att utgöra en selektiv barriär mellan den yttre och inre miljön i tarmen. Den typiska tarmskadan vid celiaki karakteriseras av avsaknad av villi och kraftigt förlängda kryptor, och både näringsupptaget och barriärfunktionen är dessutom störda. Den enda behandling som finns att tillgå vid celiaki är en livslång glutenfri diet. De skadliga proteinerna i vetegluten kallas gliadin, och det finns liknande proteiner i råg, korn, och havre. I havre kallas proteinet avenin. Möjligheten att använda havre vid celiaki har diskuterats flitigt, men numera anses det riskfritt för majoriteten av både barn och vuxna att använda havre i den glutenfria dieten. Målet med den här avhandlingen var att undersöka hur barn med celiaki reagerar på havre i kosten. Detta studerades med avseende på antikroppar mot avenin samt med en metod som mäter halten av kväveoxid- (NO-) produkter i urinen. Ett andra mål var att studera tunntarmens struktur vid olika stadier av celiaki. I den första studien undersökte vi om celiakibarn har antikroppar i serum mot avenin. Vi fann att så var fallet och att nivåerna var signifikant högre än hos friska kontrollbarn. När barnen sattes på glutenfri kost sjönk antikroppsnivåerna, för att öka igen när gluten återinfördes i kosten. Blodproverna till den här studien togs innan debatten om havre kom igång, vilket gör att vi tror att de olika dieterna även speglar ett sant intag av havre. Studien visade också att det inte var någon korsreaktion mellan antikroppar mot avenin och gliadin. Vi använde sedan vår metod för att mäta antikroppar mot avenin i en randomiserad studie där havre gavs till barn med nydiagnostiserad celiaki. Barnen fick antingen en vanlig glutenfri diet eller en med tillsats av specialhavre. Antikroppsnivåerna sjönk markant redan efter tre månader i båda grupperna, och vid studietidens slut, efter ca ett år, hade alla utom ett par patienter återfått normala nivåer. Samma barn studerades även med avseende på NO-produkter i urinen. NO är en kortlivad molekyl som fungerar som budbärare i och mellan celler, och produktionen av den ökar markant vid en inflammation. Tidigare studier har visat att barn med obehandlad celiaki har extremt höga halter av NO-produkter i urinen. I vår studie sjönk även dessa värden signifikant efter tre månader, och det var ingen skillnad mellan grupperna. Efter ett år hade dock fyra barn i havregruppen och ett barn i den grupp som fick vanlig glutenfri kost, fortfarande extremt höga nivåer av NO-produkter. Dessa båda studier styrker den kliniska uppfattningen att de flesta barn med celiaki kan tåla havre, men de visar också att man bör följa upp de celiakibarn som kompletterar sin glutenfria kost med havre eftersom vissa barn verkar ha kvarstående tecken på inflammation i tarmen. I tarmbiopsier från barn med olika stadier av celiaki studerades förekomst och lokalisering av occludin och claudiner, proteiner som är viktiga för att upprätthålla barriärfunktionen i tarmen. Vi fann ett ökat uttryck av occludin vid obehandlad celiaki, vilket vi tror speglar den ökade celldelning och de förändrade barriäregenskaper som man ser vid aktiv celiaki. Resultaten tyder även på att uttrycket av claudin 1-5 inte tycks påverkas av kosten hos barn med celiaki. / Coeliac disease (CD) is one of our most common chronic diseases in childhood. The disease causes an intense inflammation in the small intestinal mucosa after ingestion of gluten-containing cereals in genetically predisposed individuals. The mucosal lesion in CD is characterised by villous atrophy and crypt hyperplasia, and both the absorptive and the barrier functions of the enterocytes are disturbed. The treatment of CD is a life-long adherence to a gluten-free diet (GFD). The toxic fraction of wheat gluten is gliadin, and there are similar proteins in rye, barley and oats. In oats this protein is called avenin, and it is proposed to be less toxic than the others. The use of oats in CD has been debated, but it is now considered safe for the majority of both children and adults with CD. The aims of this thesis were to investigate the humoral and inflammatory reactions to oats in children with CD, and also to study the intestinal mucosa at different stages of the disease. In a retrospective study we found that children with CD had antibodies to oats avenin, and that the levels were significantly higher than in controls. The levels attenuated during GFD, and we also showed that there was no crossreactivity between antibodies to oats and gliadin. We then used our method for measuring antibodies to avenin in a randomised, double-blind trial of oats given to children with newly diagnosed CD. The children were given either a traditional GFD or a GFD supplemented with oats. There was a rapid decrease in antibody levels in both groups already after three months on diet, and at the end of the study period all but a few had normalised their levels. The same children were also studied using urinary nitric oxide (NO) products as markers for intestinal inflammation. Likewise, these values decreased significantly after three months. At the end of the study four children in the GFD-oats group and one in the standard GFD group still had extremely high concentrations of urinary NO metabolites. Taken together, these studies strengthen the clinical impression that oats can be tolerated by the majority of children with CD, but they also warrant a caution, since there seem to be children that do not tolerate oats in their diet. The structure and distribution of occludin and claudins 1-5, tight junction proteins known to play a crucial role in maintaining the barrier function, was studied in biopsy specimens from children at different stages of CD. There was an increased expression of occludin in untreated CD, which reflects the characteristics of crypt cell hyperplasia and altered barrier properties seen in active CD. The findings also indicate that gluten intake does not significantly influence the expression and distribution of claudins 1-5 in coeliac children.

Coordination is important in groupware because it helps users collaborate efficiently. However, groupware systems in which activities occur at a faster pace need faster coordination in order to keep up with the speed of the activity. Faster coordination is especially needed when actions are dependent on one another (i.e., they are tightly-coupled) and when each user can see and interact with other users actions as they occur (i.e., real time). There is little information available about this type of fast coordination (also named high-speed coordination or HSC) in groupware. In this thesis, I addressed this problem by providing a body of principles and information about high-speed coordination. This solution was achieved by creating a groupware game called RTChess and then conducting an exploratory evaluation in which high-speed coordination was investigated. The results of this evaluation show that there were small amounts of high-speed coordination in the game and that high-speed coordination was difficult to achieve. In addition, HSC was affected by five main characteristics of the groupware environment: user experience, level of awareness of the partners interactions, communication between partners, number of dependencies that affect the users interactions, and pace of activities in the system.

There are a significant number of microwave applications, including active antenna arrays, wireless communication systems, navigational applications, etc., where improvement of such qualities as manufacturing costs, size, weight, power consumption, etc. is still on the agenda of todays RF design. In order to meet these requirements, new technologies must be actively involved in fabrication of RF components with improved characteristics. One of such fabrication technologies is called LIGA, used before primarily in fluidics, photonics, bioengineering, and micromechanics, and only recently receiving growing attention in RF component fabrication. One of the RF components suffering limitations in performance due to limitations in fabrication capabilities is the compact single metal layer (SML) coupled-line 3-dB coupler, also called hybrid, required in some applications thanks to its ability to divide power equally and electrically isolate the output from the input. In todays practical edge-coupled SML coupler designs, the level of coupling is limited by the capabilities of the photolithographic process to print the coupled lines close enough for tight coupling and it is usually no tighter that 8 dB. A promising way to overcome this limitation is increasing the area of metallic interface of the coupled lines, thus increasing the mutual capacitance of the lines, and inherently the coupling between them. This should be preferably done with keeping the coupler compact with respect to the footprint area, which is attained by making taller conductors, i.e. employing the third dimension. In contrast with previously used RF component fabrication processes, LIGA is the technology that allows the designer to explore the third dimension and build tall conductors while being also able to use small features. When the two-dimensional edge-coupled SML couplers are extended into the three-dimensional structures, they rather become the side-coupled SML couplers. Tall-conductor coupled lines have been characterized in this work to reveal their dependence on their geometry and a 3-dB SML coupler with tall conductors has been developed and fabricated using LIGA at the Institute for Microstructure Technology (IMT), Karlsruhe, Germany. The simulation and measurement results demonstrate the potentially superior performance of LIGA couplers, and the promising capabilities of LIGA for fabrication of RF microstructures.

There are a significant number of microwave applications, including active antenna arrays, wireless communication systems, navigational applications, etc., where improvement of such qualities as manufacturing costs, size, weight, power consumption, etc. is still on the agenda of todays RF design. In order to meet these requirements, new technologies must be actively involved in fabrication of RF components with improved characteristics. One of such fabrication technologies is called LIGA, used before primarily in fluidics, photonics, bioengineering, and micromechanics, and only recently receiving growing attention in RF component fabrication. One of the RF components suffering limitations in performance due to limitations in fabrication capabilities is the compact single metal layer (SML) coupled-line 3-dB coupler, also called hybrid, required in some applications thanks to its ability to divide power equally and electrically isolate the output from the input. In todays practical edge-coupled SML coupler designs, the level of coupling is limited by the capabilities of the photolithographic process to print the coupled lines close enough for tight coupling and it is usually no tighter that 8 dB. A promising way to overcome this limitation is increasing the area of metallic interface of the coupled lines, thus increasing the mutual capacitance of the lines, and inherently the coupling between them. This should be preferably done with keeping the coupler compact with respect to the footprint area, which is attained by making taller conductors, i.e. employing the third dimension. In contrast with previously used RF component fabrication processes, LIGA is the technology that allows the designer to explore the third dimension and build tall conductors while being also able to use small features. When the two-dimensional edge-coupled SML couplers are extended into the three-dimensional structures, they rather become the side-coupled SML couplers. Tall-conductor coupled lines have been characterized in this work to reveal their dependence on their geometry and a 3-dB SML coupler with tall conductors has been developed and fabricated using LIGA at the Institute for Microstructure Technology (IMT), Karlsruhe, Germany. The simulation and measurement results demonstrate the potentially superior performance of LIGA couplers, and the promising capabilities of LIGA for fabrication of RF microstructures.

Airway epithelial damage is commonly found in asthma patients. Epithelial damage was investigated with special reference to contacts between epithelial cells. Eosinophils, common in allergic asthma, secrete cationic proteins, particularly major basic protein (MBP). The effect of poly-L-arginine, an analogue of MBP, on airway epithelial cells was investigated. Poly-L-arginine induced membrane damage, resulting in increased permeability, loss of cell-cell contracts (tight junctions and desmosomes) and generalized cell damage. Adhesion molecules on airway epithelial cells may be important in recruiting leukocytes. Interferon (IFN)-γ increased intracellular adhesion molecule-1 expression in airway epithelial cell lines. A combination of interleukin-4 and IFN-γ opened the tight junctions. Epithelial damage in asthma was studied at the ultrastructural level in bronchial biopsies from patients with atopic or non-atopic asthma, and healthy controls. Epithelial damage was extensive in both asthma groups. In basal and columnar cells, relative desmosome length was reduced by 30-40%. In columnar cells, half-desmosomes were noticed. Changes tended to be more extensive in atopic asthma, but there was no significant difference between the two groups. Reduced desmosomal contact may be important in the epithelial shedding observed in asthma. The contact area between columnar cells and basal lamina is relatively small in the human airway. Attachment of columnar cells to the basal lamina occurs indirectly, via desmosomal attachment to basal cells. Direct attachment of columnar cells to the basal lamina is weakened in asthmatics. Nasal polyposis is a chronic inflammatory disease often associated with asthma. An ultrastructural study showed that epithelial damage of columnar cells is more pronounced in allergic patients. The length of columnar cell desmosomes was significantly reduced in asthmatics vs. non-asthmatics, and in allergics vs. non-allergics. Cell contacts in airway epithelium in asthmatics are weakened, which may be an intrinsic feature or due to the presence of eosinophils producing toxic proteins.

Los métodos kp y tight-binding, que inicialmente fueron diseñados para predecir las propiedades del sólido extendido, han sido adaptados para describir las propiedades optoelectrónicas de nanoestructuras semiconductoras. El Hamiltoniano kp de 4 bandas para huecos y la ecuación de masa efectiva en el modelo de 1 banda para electrones se han discretizado en coordenadas cilíndricas, con el objetivo de estudiar los efectos de la aplicación de un campo magnético sobre el espectro energético de los nanocristales y las propiedades colectivas en sistemas de puntos cuánticos acoplados. Entre los resultados obtenidos cabe destacar que el acoplamiento entre nanocristales con topología de antidot provoca una importante estabilización energética de la minibanda fundamental, la cual permanece inalterada frente a la acción de un campo magnético.El modelo tight-binding de primeros vecinos que se ha implementado utiliza una base sp3s* para describir cada átomo del nanocristal. Este modelo atomista permite la descripción detallada de la estructura óptica fina de los nanocristales. Se ha evidenciado que los espectros teóricos de absorción con luz polarizada en la dirección z permiten discriminar entre geometrías que la microscopía electrónica no es capaz de discernir.

Coordination is important in groupware because it helps users collaborate efficiently. However, groupware systems in which activities occur at a faster pace need faster coordination in order to keep up with the speed of the activity. Faster coordination is especially needed when actions are dependent on one another (i.e., they are tightly-coupled) and when each user can see and interact with other users actions as they occur (i.e., real time). There is little information available about this type of fast coordination (also named high-speed coordination or HSC) in groupware. In this thesis, I addressed this problem by providing a body of principles and information about high-speed coordination. This solution was achieved by creating a groupware game called RTChess and then conducting an exploratory evaluation in which high-speed coordination was investigated. The results of this evaluation show that there were small amounts of high-speed coordination in the game and that high-speed coordination was difficult to achieve. In addition, HSC was affected by five main characteristics of the groupware environment: user experience, level of awareness of the partners interactions, communication between partners, number of dependencies that affect the users interactions, and pace of activities in the system.