Structural studies on enzymes of biotechnological and biomedical interest

Structural studies of proteins aim at elucidating the atomic details of molecular interactions in biological processes of living organisms. These studies are particularly important in understanding structure, function and evolution of proteins and in defining their roles in complex biological settings. Furthermore, structural studies can be used for the development of novel properties in biomolecules of environmental, industrial and medical importance. X-ray crystallography is an invaluable tool to obtain accurate and precise information about the structure of proteins at the atomic level. Glutathione transferases (GSTs) are amongst the most versatile enzymes in nature. They are able to catalyze a wide variety of conjugation reactions between glutathione (GSH) and non-polar components containing an electrophilic carbon, nitrogen or sulphur atom. Plant GSTs from the Tau class (a poorly characterized class) play an important role in the detoxification of xenobiotics and stress tolerance. Structural studies were performed on a Tau class fluorodifen-inducible glutathione transferase from Glycine max (GmGSTU4-4) complexed with GSH (2.7 Å) and a product analogue Nb-GSH (1.7 Å). The three-dimensional structure of the GmGSTU4-4-GSH complex revealed that GSH binds in different conformations in the two subunits of the dimer: in an ionized form in one subunit and a non-ionized form in the second subunit. Only the ionized form of the substrate may lead to the formation of a catalytically competent complex. Structural comparison between the GSH and Nb-GSH bound complexes revealed significant differences with respect to the hydrogen-bonding, electrostatic interaction pattern, the upper part of -helix H4 and the C-terminus of the enzyme. These differences indicate an intrasubunit modulation between the G-and Hsites suggesting an induced-fit mechanism of xenobiotic substrate binding. A novel binding site on the surface of the enzyme was also revealed. Bacterial type-II L-asparaginases are used in the treatment of haematopoietic diseases such as acute lymphoblastic leukaemia (ALL) and lymphomas due to their ability to catalyze the conversion of L-asparagine to L-aspartate and ammonia. Escherichia coli and Erwinia chrysanthemi asparaginases are employed for the treatment of ALL for over 30 years. However, serious side-effects affecting the liver and pancreas have been observed due to the intrinsic glutaminase activity of the administered enzymes. Structural studies on Helicobacter pylori L-asparaginase (HpA) were carried out in an effort to discover novel L-asparaginases with potential chemotherapeutic utility in ALL treatment. Detailed analysis of the active site geometry revealed structurally significant differences between HpA and other Lasparaginases that may be important for the biological activities of the enzyme and could be further exploited in protein engineering efforts.

Innovations of ageing and societal transition : Dynamics of change of the socio-technical regime of ageing

The purpose of this dissertation is to examine the dynamics of the socio-technical system in the field of ageing. The study stems from the notion that the ageing of the population as a powerful megatrend has wide societal effects, and is not just a matter for the social and health sector. The central topic in the study is change: not only the age structures and structures of society are changing, but also at the same time there is constant development, for instance, in technologies, infrastructures and cultural perceptions. The changing concept of innovation has widened the understanding of innovations related to ageing from medical and assistive technological innovations to service and social innovations, as well as systemic innovations at different levels, which means the intertwined and co-evolutionary change in technologies, structures, services and thinking models. By the same token, the perceptions of older people and old age are becoming more multi-faceted: old age is no longer equated to illnesses and decline, but visions of active ageing and a third age have emerged, which are framed by choices, opportunities, resources and consumption in later life. The research task in this study is to open up the processes and mechanisms of change in the field of ageing, which are studied as a complex, multi-level and interrelated socio-technical system. The question is about co-effective elements consisting of macro-level landscape changes, the existing socio-technical regime (the rule system, practices and structures) and bottom-up niche-innovations. Societal transitions do not account for the things inside the regime alone, or for the long-term changes in the landscape, nor for the radical innovations, but for the interplay between all these levels. The research problem is studied through five research articles, which offer micro-level case studies to macro-level phenomenon. Each of the articles focus on different aspects related to ageing and change, and utilise various datasets. The framework of this study leans on the studies of socio-technical systems and multi-level perspective on transitions mainly developed by Frank Geels. Essential factors in transition from one socio-technological regime to another are the co-evolutionary processes between landscape changes, regime level and experimental niches. Landscape level changes, like the ageing of the population, destabilise the regime in the forms of coming pressures. This destabilization offers windows for opportunity to niche-innovations outside or at fringe of the regime, which, through their breakthrough, accelerate the transition process. However, the change is not easy because of various kinds of lock-ins and inertia, which tend to maintain the stability of the regime. In this dissertation, a constructionist approach of society is applied leaning mainly to the ideas of Anthony Giddens’ theory of structuration, with the dual nature of structures. The change is taking place in the interplay between actors and structures: structures shape people’s practices, but at the same time these practices constitute and reproduce social systems. Technology and other material aspects, as part of socio-technical systems, and the use of them, also take part in the structuration process. The findings of the study point out that co-evolutionary and co-effective relationships between economic, cultural, technological and institutional fields, as well as relationships between landscape changes, changes in the local and regime-level practices and rule systems, are a very complex and multi-level dynamic socio-technical phenomenon. At the landscape level of ageing, which creates the pressures and triggers to the regime change, there are three remarkable megatrends: demographic change, changes in the global economy and the development of technologies. These exert pressures to the socio-technical regime, which as a rule system is experiencing changes in the form of new markets and consumer habits, new ways of perceiving ageing, new models of organising the health care and other services and as new ways of considering innovation and innovativeness. There are also inner dynamics in the relationships between these aspects within the regime. These are interrelated and coconstructed: the prevailing perceptions of ageing and innovation, for instance, reflect the ageing policies, innovation policies, societal structures, organising models, technology and scientific discussion, and vice versa. Technology is part of the inner dynamics of the sociotechnological regime. Physical properties of the artefacts set limitations and opportunities with regard to their functions and uses. The use of and discussion about technology, contributes producing and reproducing the perceptions of old age. For societal transition, micro-level changes are also needed, in form of niche-innovations, for instance new services, organisational models or new technologies, Regimes, as stabilitystriven systems, tend to generate incremental innovations, but radically new innovations are generated in experimental niches protected from ‘normal’ market selection. The windows of opportunity for radical novelties may be opened if the circumstances are favourable for instance by tensions in the socio-technical regime affected by landscape level changes. This dissertation indicates that a change is taking place, firstly, in the dynamic interactionbetween levels, as a result of purposive action and governance to some extent. Breaking the inertia and using the window of opportunity for change and innovation offered by dynamics between levels, presupposes the actors’ special capabilities and actions such as dynamic capabilities and distance management. Secondly, the change is taking place the socio-technological negotiations inside the regime: interaction between technological and social, which is embodied in the use of technology. The use of technology includes small-level contextual scripts that also participate in forming broader societal scripts (for instance defining old age at the society level), which in their turn affect the formation of policies for innovation and ageing. Thirdly, the change is taking place by the means of active formation of the multi-actor innovation networks, where the role of distance management is crucial to facilitate the communication between actors coming from different backgrounds as well as to help the niches born outside the regime to utilise the window of opportunity offered by regime destabilisation. This dissertation has both theoretical and practical contributions. This study participates in the discussion of action-oriented view on transition by opening up of the socio-technological, coevolutionary processes of the multi-faceted phenomenon of ageing, which has lacked systematic analyses. The focus of this study, however, is not on the large-scale coordination and governance, but rather on opening up the incremental elements and structuration processes, which contribute to the transition little by little, and which can be affected to. This increases the practical importance of this dissertation, by highlighting the importance of very tiny, everyday elements in the change processes in the long run.

Of pearls and pigs : a conceptual-semantic Tiernet approach to formal representation of structure and variation of phraseological units

"Helmiä sioille", pärlor för svin, säger man på finska om någonting bra och fint som tas emot av en mottagare som inte vill eller har ingen förmåga att förstå, uppskatta eller utnyttja hela den potential som finns hos det mottagna föremålet, är ointresserad av den eller gillar den inte. För sådana relativt stabila flerordiga uttryck, som är lagrade i språkbrukarnas minnen och som demonstrerar olika slags oregelbundna drag i sin struktur använder man inom lingvistiken bl.a. termerna "idiom" eller "fraseologiska enheter". Som en oregelbundenhet kan man t.ex. beskriva det faktum att betydelsen hos uttrycket inte är densamma som man skulle komma till ifall man betraktade det som en vanlig regelbunden fras. En annan oregelbundenhet, som idiomforskare har observerat, ligger i den begränsade förmågan att varieras i form och betydelse, som många idiom har jämfört med regelbundna fraser. Därför talas det ofta om "grundform" och "grundbetydelse" hos idiom och variationen avses som avvikelse från dessa. Men när man tittar på ett stort antal förekomstexempel av idiom i språkbruk, märker man att många av dem tillåter variation, t.o.m. i sådan utsträckning att gränserna mellan en variant och en "grundform" suddas ut, och istället för ett idiom råkar vi plötsligt på en "familj" av flera besläktade uttryck. Allt detta väcker frågan om hur dessa uttryck egentligen ska vara representerade i språket. I avhandlingen utförs en kritisk granskning av olika tidigare tillvägagångssätt att beskriva fraseologiska enheter i syfte att klargöra vilka svårigheter deras struktur och variation erbjuder för den lingvistiska teorin. Samtidigt presenteras ett alternativt sätt att beskriva dessa uttryck. En systematisk och formell modell som utvecklas i denna avhandling integrerar en beskrivning av idiom på många olika språkliga nivåer och skildrar deras variation i form av ett nätverk och som ett resultat av samspel mellan idiomets struktur och kontexter där det förekommer, samt av interaktion med andra fasta uttryck. Modellen bygger på en fördjupande, språkbrukbaserad analys av det finska idiomet "X HEITTÄÄ HELMIÄ SIOILLE" (X kastar pärlor för svin).

Boron influence on concentration of polyols and other sugars in Eucalyptus

Although the functions of the element Boron (B) in plants have not been sufficiently clarified, several hypotheses have been raised. Some of the functions attributed to this element are synthesis and transport of carbohydrates. To evaluate the effect of B on the synthesis of some polyols and sugars in Eucalyptus grandis and hybrids "urograndis", these species were submitted to situations of supply and restriction of B. The experiment was carried out in a greenhouse, arranged in a randomized block design in a 2 x 2 factorial scheme, with 5 replicates of each genotype. The B levels were provided in the form of nutrient solution. No significant difference was observed in the content of mannitol, sorbitol, myo-inositol and scyllo-inositol and sugars α-glucose and β-glucose between E. grandis and hybrids. Arabinose was the only one to present a higher content in E. grandis on restriction of B. The effect of the presence of B was very expressive, but regarding B supply, the plants showed significant increase in the synthesis of the compounds evaluated.

Professional development among educators pursuing a B.Ed. program in special education in Tanzania : a study of motives and perceived outcomes

Implementation of different policies and plans aiming at providing education for all is a challenge in Tanzania. The need for educators and professionals with relevant knowledge and qualifications in special education is substantial. Teacher education does not equip educators with sufficient knowledge and skills in special education and professional development programs in special education are few in number. Up to 2005 no degree programs in special education at university level were available in Tanzania. The B.Ed. Special Education program offered by the Open University of Tanzania in collaboration with Åbo Akademi University in Finland was one of the efforts aimed at addressing the big national need for teachers and other professionals with degree qualifications in special education. This pilot program offered unique possibilities to study professional development in Tanzania. The research group in this study consisted of the group of students who participated in the degree program 2005-2007. The study is guided by three theoretical perspectives: individual, social and societal. The individual perspective emphasizes psychological factors as motives, motivation, achievement, self-directed behavior and personal growth. Within social perspective, professional development is viewed as situated within the social and cultural context. The third perspective, the societal, focuses on change, reforms, innovations and transformation of school systems and societies. Accordingly, professional development is viewed as an individual, social and societal phenomenon. The overall aim of the study is to explore the participants’ motives for participating in a B.Ed. Special Education program and the perceived outcomes of the program in terms of professional development. In order to achieve the objectives of the study, a case study approach was adopted. Questionnaires and semi-structured interviews were administered in three waves between January 2007 and February 2009 to the 35 educators participating in the B.Ed. Special Education program. The findings of the study reveal that the participants expressed motives which were related to job performance, knowledge, skills, academic degree and career. Also altruistic motives were expressed by the participants in terms of helping and supporting students with special needs and their communities. The perceived outcomes of the program were in line with the expressed motives. However, the results indicate that the participants also learned new skills, as interaction skills and guidance and counseling skills. Increased self-confidence was also mentioned as an outcome. The participants also got deepened understanding of disability issues. In addition, they learned strategies for creating awareness of persons with disability in the communities. Thus the findings of the study indicate positive outcomes of the program in terms of professional development. The conclusion of the study is that individual, social and societal factors interact when it comes to explaining why Tanzanian educators in special education choose to pursue a degree program in special education. The individual motives, as increased knowledge and better prospects of career development interact with the social and societal motives to help and support vulnerable student groups. The study contributes to increased understanding of the complexity of professional development and of the realities educators meet when educational reforms are implemented in a developing country.

Outbreaks of rhinofacial and rhinopharyngeal zygomycosis in sheep in Paraíba, northeastern Brazil

Two outbreaks of zigomycosis with rhinofacial and two other with rhinopharyngeal lesions involving fungi with filamentous coaenocytic hyphae characteristic of entomoph-thoramycetous fungi are reported in the state of Paraíba, northeastern Brazil. One outbreak of rhinofacial zygomycosis occurred during the rainy season affecting 5 sheep. Another outbreak of the clinical form affected one out of 40 sheep during the dry season. Common clinical signs of the rhinofacial infection were bilateral serosanguineous nasal discharge with swelling of nostrils, upper lip, and the skin of the face. At necropsy the nasal mucosa showed dark brownish ulcerated areas which extended from the mucocutaneous region to 10cm inside the nasal vestibule. The mucosa of the hard palate was also ulcerated. The cutting surface of nostrils and palate showed a brownish or red spongeous tissue of friable consistency. One outbreak of rhinopharyngitis took place on an irrigated coconut farm; 7 out of 60 adult sheep were affected. Another outbreak affected a sheep in a flock of 80 during the dry season. Clinical signs as noisy respiration and dyspnoea due to mechanical blockage of the nasal cavities, swelling of the nostrils, and serosanguineous nasal discharge were observed. Six out of 8 sheep in this group showed exophthalmia, keratitis and unilateral corneal ulceration of the eye. The sheep either died of their infection or were euthanized after a clinical course of 7-30 days. At necropsy there was a dense yellow exudate in the nasopharyngeal area affecting the ethmoidal region, turbinate bones, paranasal sinuses, hard and soft palates, orbital cavity, pharynges, regional muscles and lymph nodes. Histopathologically both forms of the disease showed multifocal granulomas with an eosinophilic necrotic reaction (Splendore-Hoeppli phenomenon) containing ribbon-type coenocytic hyphae with 7-30mum in diameter similar to hyphae of zygomycetous fungi, possibly Conidiobolus spp. Outbreaks of both forms of mycotic rhinitis are common in northeastern Brazil and in other regions of the country.

Immunohistochemical study of genital and extragenital forms of canine transmissible venereal tumor in Brazil

Aiming to provide insight and discussing the problems related to the diagnosis and differential diagnosis of canine transmissible venereal tumor (CTVT), especially in its extragenital form, immunohistochemical evaluation was performed and a comparison was established by analysis of the microscopic appearance of 10 genital CTVTs and 13 exclusively extragenital CTVTs previously diagnosed by cytology and histopathology. CTVTs samples were incubated with biotinylated antibodies raised against specific membrane (anti-macrophage) and cytoplasmic antigens (anti-lysozyme, anti-S-100 protein, anti-vimentin and anti-CD18) and subsequently developed using streptavidin-biotin peroxidase and streptavidin-biotin-alkaline phosphatase methods. A strong reactivity with the anti-vimentin antibody was found in 100% of the tumors tested (22/22). No reactivity was found for the anti-lysozyme, anti-macrophage, anti-S-100 protein and anti-CD18. No histopathological or immunoreactivity differences between genital and extragenital CTVTs were found. These findings do not corroborate the hypothesis of histiocytic origin of CTVT (no reactivity to anti-lysozyme, anti-macrophage and anti-CD 18 antibodies). In addition, the antibody panel used is useful to narrow the differential diagnosis for lymphomas, histiocytic tumors, amelanotic melanomas, and poorly differentiated epithelial neoplasias, among others.

The accumulation of financial and social capital as a means to achieve a sustained competitive advantage for consumer co-operatives

By so far, scholars have discussed how the characteristics of consumer co-operatives (cooperative principles, values and the dual role of members as the users and owners) can potentially give them a competitive advantage over investor-owned firms (IOFs). In addition, concern for the community (as partly derived from locality and regionality) has been seen as a potential source of success for consumer co-operatives. On the other hand, the geographicbound purpose of consumer co-operation causes that consumer co-operative can be regarded as a challenging company form to manage. This is because, according to the purpose of consumer co-operation, co-operatives are obligated to 1) provide the owners with services and goods that are needed and do so at more affordable prices than their competitors do and/or 2) to operate in areas in which competitors do not want to operate (for example, because of the low profitability in certain area of business or region). Thus, consumer co-operatives have to operate very efficiently in order to execute this geographic-bound corporate purpose (e.g. they cannot withdraw from the competition during the declining stages of business). However, this efficiency cannot be achieved by any means; as the acceptance from the important regional stakeholders is the basic operational precondition and lifeline in the long run. Thereby, the central question for the survival and success of consumer co-operatives is; how should the consumer co-operatives execute its corporate purpose so it can be the best alternative to its members in the long run? This question has remained unanswered and lack empirical evidence in the previous studies on the strategic management of consumer cooperation. In more detail, scholars have not yet empirically investigated the question: How can consumer co-operatives use financial and social capital to achieve a sustained competitive advantage? It is this research gap that this doctoral dissertation aims to fulfil. This doctoral dissertation aims to answer the above questions by combining and utilizing interview data from S Group co-operatives and the central organizations in S Group´s network (overall, 33 interviews were gathered), archival material and 56 published media articles/reports. The study is based on a qualitative case study approach that is aimed at theory development, not theory verification (as the theory is considered as nascent in this field of study). Firstly, the findings of this study indicate that consumer co-operatives accumulate financial capital; 1) by making profit (to invest and grow) and 2) by utilizing a network-based organizational structure (local supply chain economies). As a result of financial capital accumulation, consumer co-operatives are able to achieve efficiency gains but also remain local. In addition, a strong financial capital base increases consumer co-operatives´ independence, competitiveness and their ability to participate in regional development (which is in accordance with their geographically bound corporate purpose). Secondly, consumer cooperatives accumulate social capital through informal networking (with important regional stakeholders), corporate social responsibility (CSR) behaviour and CSR reporting, pursuing common good, and interacting and identity sharing. As a result of social capital accumulation, consumer co-operatives are able to obtain the resources for managing; 1) institutional dependencies and 2) customer relations. By accumulating both social and financial capital through the above presented actions, consumer co-operatives are able to achieve sustained competitive advantage. Finally, this thesis provides useful ideas and new knowledge for cooperative managers concerning why and how consumer co-operatives should accumulate financial and social capital (to achieve sustained competitive advantage), while aligning with their corporate purpose.

Of milquetoasts and daredevils : personalities in female eiders

Traditionally biologists have often considered individual differences in behaviour or physiology as a nuisance when investigating a population of individuals. These differences have mostly been dismissed as measurement errors or as non-adaptive variation around an adaptive population mean. Recent research, however, challenges this view. While long acknowledged in human personality studies, the importance of individual variation has recently entered into ecological and evolutionary studies in the form of animal personality. The concept of animal personality focuses on consistent differences within and between individuals in behavioural and physiological traits across time and contexts and its ecological and evolutionary consequences. Nevertheless, a satisfactory explanation for the existence of personality is still lacking. Although there is a growing number of explanatory theoretical models, there is still a lack of empirical studies on wild populations showing how traditional life-history tradeoffs can explain the maintenance of variation in personality traits. In this thesis, I first investigate the validity of variation in allostatic load or baseline corticosterone (CORT) concentrations as a measure for differences in individual quality. The association between CORT and quality has recently been summarised under the “CORT-fitness hypothesis”, which states that a general negative relationship between baseline CORT and fitness exists. I then continue to apply the concept of animal personality to depict how the life-history trade-off between survival and fecundity is mediated in incubating female eiders (Somateria mollissima), thereby maintaining variation in behaviour and physiology. To this end, I investigated breeding female eiders from a wild population that breeds in the archipelago around Tvärminne Zoological Station, SW Finland. The field data used was collected from 2008 to 2012. The overall aim of the thesis was to show how differences in personality and stress responsiveness are linked to a life-history context. In the four chapters I examine how the life-history trade-off between survival and fecundity could be resolved depending on consistent individual differences in escape behaviour, stress physiology, individual quality and nest-site selection. First, I corroborated the validity of the “CORT-fitness hypothesis”, by showing that reproductive success is generally negatively correlated with serum and faecal baseline CORT levels. The association between individual quality and baseline CORT is, however, context dependent. Poor body condition was associated with elevated serum baseline CORT only in older breeders, while a larger reproductive investment (clutch mass) was associated with elevated serum baseline CORT among females breeding late in the season. Interestingly, good body condition was associated with elevated faecal baseline CORT levels in late breeders. High faecal baseline CORT levels were positively related to high baseline body temperature, and breeders in poor condition showed an elevated baseline body temperature, but only on open islands. The relationship between stress physiology and individual quality is modulated by breeding experience and breeding phenology. Consequently, the context dependency highlights that this relationship has to be interpreted cautiously. Additionally, I verified if stress responsiveness is related to risk-taking behaviour. Females who took fewer risks (longer flight initiation distance) showed a stronger stress response (measured as an increase in CORT concentration after capture and handling of the bird). However, this association was modulated by breeding experience and body condition, with young breeders and those in poor body condition showing the strongest relationship between risktaking and stress responsiveness. Shy females (longer flight initiation distance) also incubated their clutch for a shorter time. Additionally, I demonstrated that stress responsiveness and predation risk interact with maternal investment and reproductive success. Under high risk of predation, females that incubated a larger clutch showed a stronger stress response. Surprisingly, these females also exhibited higher reproductive success than females with a weaker stress response. Again, these context dependent results suggest that the relationship between stress responsiveness and risk-taking behaviour should not be studied in isolation from individual quality and that stress responsiveness may show adaptive plasticity when individuals are exposed to different predation regimes. Finally, female risk-taking behaviour and stress coping styles were also related to nest-site choice. Less stress responsive females more frequently occupied nests with greater coverage that were farther away from the shoreline. Females nesting in nests with medium cover and farther from the shoreline had higher reproductive success. These results suggest that different personality types are distributed non-randomly in space. In this thesis I was able to demonstrate that personalities and stress coping strategies are persistent individual characteristics, which express measurable effects on fitness. This suggests that those traits are exposed to natural selection and thereby can evolve. Furthermore, individual variation in personality and stress coping strategy is linked to the alternative ways in which animals resolve essential life-history trade-offs.

Mathematics inspired by Darwin. Adaptive dynamics of dispersal and cooperation

In 1859, Charles Darwin published his theory of evolution by natural selection, the process occurring based on fitness benefits and fitness costs at the individual level. Traditionally, evolution has been investigated by biologists, but it has induced mathematical approaches, too. For example, adaptive dynamics has proven to be a very applicable framework to the purpose. Its core concept is the invasion fitness, the sign of which tells whether a mutant phenotype can invade the prevalent phenotype. In this thesis, four real-world applications on evolutionary questions are provided. Inspiration for the first two studies arose from a cold-adapted species, American pika. First, it is studied how the global climate change may affect the evolution of dispersal and viability of pika metapopulations. Based on the results gained here, it is shown that the evolution of dispersal can result in extinction and indeed, evolution of dispersalshould be incorporated into the viability analysis of species living in fragmented habitats. The second study is focused on the evolution of densitydependent dispersal in metapopulations with small habitat patches. It resulted a very surprising unintuitive evolutionary phenomenon, how a non-monotone density-dependent dispersal may evolve. Cooperation is surprisingly common in many levels of life, despite of its obvious vulnerability to selfish cheating. This motivated two applications. First, it is shown that density-dependent cooperative investment can evolve to have a qualitatively different, monotone or non-monotone, form depending on modelling details. The last study investigates the evolution of investing into two public-goods resources. The results suggest one general path by which labour division can arise via evolutionary branching. In addition to applications, two novel methodological derivations of fitness measures in structured metapopulations are given.

Anatomical and ultrastructural aspects of root and mycorrhiza of Anadenanthera peregrina (L.) Speg. var. falcata (Benth.) Altschul (Leguminosae-Mimosoideae)

A. peregrina var. falcata form mutualistic symbiosis with arbuscular mycorrhizal fungus. An anatomical and ultrastructural study was carried out to analyze some aspects of this simbiotic association as well as some root features. The results evidenced the presence of fibers with non-lignified thicked secondary walls in the stele and sparse papillae on root surface. A. peregrina var. falcata mycorrhizas presented features of Arum-type (intercellular hyphae) and Paris-type (extensive coils) arbuscular mycorrhiza. Their general appearance with extraradical hyphae, intracellular coils, intercellular hyphae and arbuscules, is in agreement with arbuscular mycorrhizas of several plants. The ultrastructural observations showed that in intercellular hyphae and arbuscules vacuoles were dominant and that in rough endoplasmatic reticulum and small vesicles seems to be associated with arbuscule senescence process.

The interplay of JNK and SCG10 during cortical development and in excitotoxic responses in brain

Initially identified as stress activated protein kinases (SAPKs), the c-Jun Nterminal kinases (JNKs) are currently accepted as potent regulators of various physiologically important cellular events. Named after their competence to phosphorylate transcription factor c-Jun in response to UVtreatment, JNKs play a key role in cell proliferation, cell death or cell migration. Interestingly, these functions are crucial for proper brain formation. The family consists of three JNK isoforms, JNK1, JNK2 and JNK3. Unlike brain specific JNK3 isoform, JNK1 and JNK2 are ubiquitously expressed. It is estimated that ten splice variants exist. However, the detailed cellular functions of these remain undetermined. In addition, physiological conditions keep the activities of JNK2 and JNK3 low in comparison with JNK1, whereas cellular stress raises the activity of these isoforms dramatically. Importantly, JNK1 activity is constitutively high in neurons, yet it does not stimulate cell death. This suggests a valuable role for JNK1 in brain development, but also as an important mediator of cell wellbeing. The aim of this thesis was to characterize the functional relationship between JNK1 and SCG10. We found that SCG10 is a bona fide target for JNK. By employing differential centrifugation we showed that SCG10 co-localized with active JNK, MKK7 and JIP1 in a fraction containing endosomes and Golgi vesicles. Investigation of JNK knockout tissues using phosphospecific antibodies recognizing JNK-specific phosphorylation sites on SCG10 (Ser 62/Ser 73) showed that phosphorylation of endogenous SCG10 was dramatically decreased in Jnk1-/- brains. Moreover, we found that JNK and SCG10 co-express during early embryonic days in brain regions that undergo extensive neuronal migration. Our study revealed that selective inhibition of JNK in the cytoplasm significantly increased both the frequency of exit from the multipolar stage and radial migration rate. However, as a consequence, it led to ill-defined cellular organization. Furthermore, we found that multipolar exit and radial migration in Jnk1 deficient mice can be connected to changes in phosphorylation state of SCG10. Also, the expression of a pseudo-phosphorylated mutant form of SCG10, mimicking the JNK1- phopshorylated form, brings migration rate back to normal in Jnk1 knockout mouse embryos. Furthermore, we investigated the role of SCG10 and JNK in regulation of Golgi apparatus (GA) biogenesis and whether pathological JNK action could be discernible by its deregulation. We found that SCG10 maintains GA integrity as with the absence of SCG10 neurons present more compact fragmented GA structure, as shown by the knockdown approach. Interestingly, neurons isolated from Jnk1-/- mice show similar characteristics. Block of ER to GA is believed to be involved in development of Parkinson's disease. Hence, by using a pharmacological approach (Brefeldin A treatment), we showed that GA recovery is delayed upon removal of the drug in Jnk1-/- neurons to an extent similar to the shRNA SCG10-treated cells. Finally, we investigated the role of the JNK1-SCG10 duo in the maintenance of GA biogenesis following excitotoxic insult. Although the GA underwent fragmentation in response to NMDA treatment, we observed a substantial delay in GA disintegration in neurons lacking either JNK1 or SCG10.

The effect of methotrexate and azathioprine on the serum levels of IgA-a1-antitrypsin complex in juvenile chronic arthritis

In the present study we investigated the influence of methotrexate (MTX) and azathioprine (AZA) on the serum levels of the IgA-a1-antitrypsin (IgA-AT) complex in patients with the systemic form of juvenile chronic arthritis (JCA). Fifty-six JCA patients (22 treated with MTX, 18 treated with AZA, and 16 not treated with any immunosuppressive agent) were enrolled in the study. MTX dosage ranged from 0.3 to 0.5 mg kg-1 week-1, while AZA was given daily at an average dose of 1 mg/kg. MTX was given for 13 months (SD = 7 months) whereas AZA for 11 months (SD = 6 months). The average value of the complex was higher in JCA patients than in both control groups (0.74 ± 0.73 U vs 0.37 ± 0.13 U (control children), P<0.001 and vs 0.23 ± 0.12 U (control adults), P<0.001). Values exceeding the normal range were found in twenty-two JCA patients (39.4%). Serum IgA-AT level was lowest in the MTX group compared to AZA and non-treated patients (0.56 ± 0.24 U, 0.76 ± 0.43 U, 0.95 ± 0.52 U, respectively, P<0.05). IgA values exceeding normal levels for age were found in 14% of the patients. A correlation between the levels of the IgA-AT complex and C-reactive protein (r = 0.43, P<0.01), a1-acid-glycoprotein (r = 0.45, P<0.01), a1-antichymotrypsin (r = 0.52, P<0.01), a1-antitrypsin (r = 0.40, P<0.01) and IgA (r = 0.56, P<0.01) was established

Autoradiography reveals regional metabolic differences in the endometrium of pregnant and nonpregnant mice

The rodent endometrium undergoes remarkable modifications during pregnancy, resulting from a redifferentiation of its fibroblasts. During this modification (decidualization), the fibroblasts transform into large, polyhedral cells that establish intercellular junctions. Decidualization proceeds from the subepithelial stroma towards the deep stroma situated next to the myometrium and creates regions composed of cells in different stages of differentiation. We studied by autoradiography whether cells of these different regions have different levels of macromolecular synthesis. Radioactive amino acids or radioactive sulfate were administered to mice during estrus or on different days of pregnancy. The animals were killed 30 min after injection of the precursors and the uteri were processed for light microscope autoradiography. Silver grains were counted over cells of different regions of the endometrium and are reported as the number of silver grains per area. Higher levels of incorporation of amino acids were found in pregnant animals as compared to animals in estrus. In pregnant animals, the region of decidual cells or the region of fibroblasts transforming into decidual cells showed the highest levels of synthesis. Radioactive sulfate incorporation, on the other hand, was generally higher in nonpregnant animals. Animals without decidual cell transformation (nonpregnant and 4th day of pregnancy) showed a differential incorporation by subepithelial and deep stroma fibroblasts. This study shows that regional differences in synthetic activity exist in cells that are in different stages of transformation into decidual cells as well as in different regions of the endometrium of nonpregnant mice

Studies on membrane properties of cholesterol and 3-beta modified sterol analogs

Cholesterol (Chol) is an important lipid in cellular membranes functioning both as a membrane fluidity regulator, permeability regulator and co-factor for some membrane proteins, e.g. G-protein coupled receptors. It also participates in the formation of signaling platforms and gives the membrane more mechanical strenght to prevent osmotic lysis of the cell. The sterol structure is very conserved and already minor structural modifications can completely abolish its membrane functions. The right interaction with adjacent lipids and the preference of certain lipid structures over others are also key factors in determining the membrane properties of cholesterol. Because of the many important properties of cholesterol it is of value to understand the forces and structural properties that govern the membrane behavior of this sterol. In this thesis we have used established fluorescence spectroscopy methods to study the membrane behavior of both cholesterol and some of its 3β-modified analogs. Using several fluorescent probes we have established how the acyl chain order of the two main lipid species, sphingomyelin (SM) and phosphatidylcholine (PC) affect sterol partitioning as well as characterized the membrane properties of 3β-aminocholesterol and cholesteryl phosphocholine. We concluded that cholesterol prefers SM over PC at equal acyl chain order, indicating that other structural properties besides the acyl chain order are important for sphingomyelin-sterol interactions. A positive charge at the 3β position only caused minor changes in the sterol membrane behavior compared to cholesterol. A large phosphocholine head group caused a disruption in membrane packing together with other membrane lipids with large head groups, but was also able to form stable fluid bilayers together with ceramide and cholesterol. The Ability of the large head group sterol to form bilayers together with ceramide was further explored in the last paper where cholesteryl phosphocholine/ceramide (Chol-PC/Cer) complexes were successfully used to transfer ceramide into cultured cells.