Rethinking connectedness : an investigation into the access of teacher professional learning in regional and remote Western Australia

Many teachers working in remote and regional areas have limited access to collegial support networks. This research aimed to examine the existing strategies that were being undertaken by the Department of Education in Western Australia, to provide professional learning to teachers in regional and remote areas. It was important to establish the perceptions of teachers’ access to professional learning from those working at the coalface in geographically dispersed areas. Consequently, the possible opportunity for improving the amount and variety of professional learning, through the application of both synchronous and asynchronous technologies was proposed. The study was guided by the primary research question: “In what ways might technology be used to support professional development of regional and remote teachers in Western Australia?” Generating descriptions of current practice of professional learning along with the teacher perceptions were central to this research endeavour. The study relied on a mixed method research approach in order to attend to the research question. The data were collected in phases, referred to as an explanatory mixed methods design. Quantitative data were collected from 104 participants to provide a general picture of the research problem. To further refine this general picture, qualitative data were collected through interviews and e-interviews of 10 teachers. Participants in the study included graduate teachers, teachers who had taught more than two years, senior teachers and Level Three teachers from seven teaching districts within Western Australia. An investigation into current practice was included in this phase and technologies available to support a professional learning community over distance were documented. The final phase incorporated the formulation of a conceptual framework where a model was developed to facilitate the successful implementation of a professional learning community through the application of synchronous and asynchronous technologies. The study has identified that travel time in order to access professional development is significant and impacts on teachers’ personal time. There are limited relief teachers available in these isolated areas which impacts on the opportunities to access professional development. Teachers face inequities, in terms of promotion, because professional development is explicitly linked to promotional opportunities. Importantly, it was found that professional learning communities are valued, but are often limited by small staff numbers at the geographic locality of the school. Teachers preferred to undertake professional learning in the local context of their district, school or classroom and this professional learning must be established at the need of the individual teacher in line with the school priorities. Teachers reported they were confident in using technology and accessing professional development online if required, however, much uncertainty surrounded the use of web 2.0 technologies for this purpose. The recommendations made from the study are intended to identify how a professional learning community might be enhanced through synchronous and asynchronous technologies.

Teachers working in an online world

This book brings together a number of academics who have conducted research and written about effective practices and pedagogies that incorporate the use of information and communications technologies (ICT). The book is intended for graduate and undergraduate students in Teacher Education programmes, as well as teachers and those who areinterested in contemporary educational issues. The authors in this book have been engaged in rethinking education with ICT. Implicit in this, is the view that we need to reconceptualise our pedagogies and practices in order to make schools relevant to the lives of the young people who inhabit them. The chapters in this book are based on empirically grounded research work. The chapters illustrate the various dimensions of innovative practices with ICT that can extend teachers’ pedagogies and engage learners so that they are able to extend their potential for knowledge building in new and dynamic ways.

Tibetan Market Participation in China

Since the Chinese government began implementing economic reforms in the late 1970s, China has experienced profound economic change and growth. Like other parts of China, Tibetan areas of China have also experienced wide-ranging economic change with growth even higher than the China-wide average in certain years. Though China s strategic policy of developing the West provided many opportunities for economic and business activities, Tibetans have proven poorly equipped to respond to and take advantage of these opportunities. This study is about people, about market participation and specifically about why Tibetans do not effectively participate in the market in the context of China s economic development process. Many political, social, cultural and environmental factors explain the difficulties met by Tibetan communities. However, this study focuses on three factors: the social and culture context, government policy and education. The Buddhistic nature of Tibetan communities, particularly the political and economic system in traditional Tibetan society, explains this, especially after implementation of new national economic policies. An inclusive economic development policy that promotes local people s participation in the market demands serious consideration of local conditions. Unfortunately, such considerations often ignore local Tibetan realities. The economic development policy in Tibetan areas in China is nearly always an attempt to replicate the inland model and open up markets, even though economic and sociopolitical conditions in Tibet are markedly unlike much of China. A consequence of these policies is increasing numbers of non-Tibetan migrants flowing into Tibetan areas with the ensuing marginalization of Tibetans in the marketplace. Poor quality education is another factor contributing to Tibetan inability to effectively participate in the market. Vocational and business education targeting Tibetans is of very low quality and reflective of government failing to consider local circumstances when implementing education policy. The relatively few Tibetans who do receive education are nearly always unable to compete with non-Tibetan migrants in commercial activity. Encouraging and promoting Tibetan participation in business development and access to quality education are crucial for a sustainable and prosperous society in the long term. Particularly, a localized development policy that considers local environmental conditions and production as well as local culture is crucial. Tibet s economic development should be based on local environmental and production conditions, while utilizing Tibetan culture for the benefit of creating a sustainable economy. Such a localized approach best promotes Tibetan market participation. Keywords: Tibet cultural policy education market participation

Enzymes with radical tendencies: the PFL family

The first glycyl radical in an enzyme was described 20 years ago and since then the family of glycyl radical enzymes (GREs) has expanded to include enzymes catalysing five chemically distinct reactions. The type enzymes of the family, anaerobic ribonucleotide reductase (RNRIII) and pyruvate formate lyase (PFL) had been studied long before it was known that they are GREs. Spectroscopic measurements on the radical and an observation that exposure to oxygen irreversibly inactivates the enzymes by cleavage of the protein proved that the radical is located on a particular glycine residue, close to the C-terminus of the protein. Both anaerobic RNRIII and PFL, are important for many anaerobic and facultative anaerobic bacteria as RNRIII is responsible for the synthesis of DNA precursors and PFL catalyses a key metabolic reaction in glycolysis. The crystal structures of both were solved in 1999 and they revealed that, although the enzymes do not share significant sequence identity, they share a similar structure - the radical site and residues necessary for catalysis are buried inside a ten stranded $\ualpha $/$\ubeta $-barrel. GREs are synthesised in an inactive form and are post-translationally activated by an activating enzyme which uses S-adenosyl methionine and an iron-sulphur cluster to generate the radical. One of the goals of this thesis work was to crystallise the activating enzyme of PFL. This task is challenging as, like GREs, the activating component is inactivated by oxygen. The experiments were therefore carried out in an oxygen free atmosphere. This is the first report of a crystalline GRE activating enzyme. Recently several new GREs have been characterised, all sharing sequence similarity to PFL but not to RNRIII. Also, the genome sequencing projects have identified many PFL-like GREs of unknown function, usually annotated as PFLs. In the present thesis I describe the grouping of these PFL family enzymes based on the sequence similarity and analyse the conservation patterns when compared to the structure of E. coli PFL. Based on this information an activation route is proposed. I also report a crystal structure of one of the PFL-like enzymes with unknown function, PFL2 from Archaeoglobus fulgidus. As A. fulgidus is a hyperthermophilic organism, possible mechanisms stabilising the structure are discussed. The organisation of an active site of PFL2 suggests that the enzyme may be a dehydratase. Keywords: glycyl radical, enzyme, pyruvate formate lyase, x-ray crystallography, bioinformatics

The university and the knowledge network: A new educational model for 21st century learning and employability

The higher education sector is under ongoing pressure to demonstrate quality and efficacy of educational provision, including graduate outcomes. Preparing students as far as possible for the world of professional work has become one of the central tasks of contemporary universities. This challenging task continues to receive significant attention by policy makers and scholars, in the broader contexts of widespread labour market uncertainty and massification of the higher education system (Tomlinson, 2012). In contrast to the previous era of the university, in which ongoing professional employment was virtually guaranteed to university-qualified individuals, contemporary graduates must now be proactive and flexible. They must adapt to a job market that may not accept them immediately, and has continually shifting requirements (Clarke, 2008). The saying goes that rather than seeking security in employment, graduates must now “seek security in employability”. However, as I will argue in this chapter, the current curricular and pedagogic approaches universities adopt, and indeed the core structural characteristics of university-based education, militate against the development of the capabilities that graduates require now and into the future.

Complimentary collaborations: Teachers and researchers co-developing best practices in art education

Australia is currently experiencing a huge cultural shift as it moves from a State-based curriculum, to a national education system. The Australian State-based bodies that currently manage teacher registration, teacher education course accreditation, curriculum frameworks and syllabi are often complex organisations that hold conflicting ideologies about education and teaching. The development of a centralised system, complete with a single accreditation body and a national curriculum can be seen as a reaction to this complexity. At the time of writing, the Australian Curriculum is being rolled out in staggered phases across the states and territories of Australia. Phase one has been implemented, introducing English, Mathematics, History and Science. Subsequent phases (Humanities and Social Sciences, the Arts, Technologies, Health and Physical Education, Languages, and year 9-10 work studies) are intended to follow. Forcing an educational shift of this magnitude is no simple task; not least because the States and Territories have and continue to demonstrate varying levels of resistance to winding down their own curricula in favour of new content with its unfamiliar expectations and organisations. The full implementation process is currently far from over, and far from being fully resolved. The Federal Government has initiated a number of strategies to progress the implementation, such as the development of the Australian Institute for Teaching and School Leadership (AITSL) to aid professional educators to implement the new curriculum. AITSL worked with professional and peak specialist bodies to develop Illustrations of Practice (hereafter IoP) for teachers to access and utilise. This paper tells of the building of one IoP, where a graduate teacher and a university lecturer collaborated to construct ideas and strategies to deliver visual arts lessons to early childhood students in a low Socio- Economic Status [SES] regional setting and discusses the experience in terms of its potential for professional learning in art education.

GDP-L-fucose: synthesis and role in inflammation

GDP-L-fucose: synthesis and role in inflammation The migration of leukocytes from intravascular locations to extravascular sites is essential to the immune responses. The initial attachment of leukocytes to the endothelium and the rolling step of the leukocyte extravasation cascade are mediated by selectins, a family of cell adhesion molecules on cell surfaces. Selectins are able to recognize glycoproteins and glycolipids containing the tetrasaccharide sialyl Lewis x (sLex, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc). Several glycosyltransferases are involved in the biosynthesis of sLex, fucosyltransferase VII (Fuc-TVII) being the last enzyme to modify the sLex structure. Fuc-TVII transfers L-fucose from GDP-L-fucose to sialylated N-acetyllactosamine. GDP-L-fucose is synthesized in the cytosol via two different metabolic pathways. The major, constitutively active de novo pathway involves conversion of GDP-α-D-mannose to GDP-β-L-fucose. In the alternative salvage pathway, L-fucokinase synthesizes from free fucose L-fucose-1-phosphate, which is further converted to GDP-L-fucose by GDP-L-fucose pyrophosphorylase. GDP-L-fucose is translocated from the cytosol to Golgi for fucosylation via the GDP-fucose transporter. This thesis involved the study of the synthesis of GDP-L-fucose via the salvage pathway: cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. The gene expression levels of these enzymes were found to be relatively high in various tissues; the mRNA levels were highest in brain, ovary and testis. This study also describes molecular cloning of rat fucosyltransferase VII (FUT7) and its expression as a functional enzyme. Gene expression levels of GDP-L-fucose synthesizing enzymes, GDP-fucose transporter and FUT7 were determined in inflamed tissues as well as cancer cells. Our results revealed a clear upregulation of the enzymes involved in the synthesis of GDP-L-fucose via de novo pathway, GDP-fucose transporter and FUT7 in inflamed tissues and in cancer cells. On the contrary, the GDP-L-fucose salvage pathway was found to be irrelevant in inflammation and in tumorigenesis. Furthermore, our results indicated the transcriptional coregulation of Golgi transporters involved in the synthesis of sulfo sLex, i.e. CMP-sialic acid, GDP-fucose and 3 phosphoadenosine 5 -phosphosulfate transporters, in inflammation.

Mu in vitro Transposition Technology in Functional Genetics and Genomics: Applications on Mouse and Bacteriophages

Transposons are mobile elements of genetic material that are able to move in the genomes of their host organisms using a special form of recombination called transposition. Bacteriophage Mu was the first transposon for which a cell-free in vitro transposition reaction was developed. Subsequently, the reaction has been refined and the minimal Mu in vitro reaction is useful in the generation of comprehensive libraries of mutant DNA molecules that can be used in a variety of applications. To date, the functional genetics applications of Mu in vitro technology have been subjected to either plasmids or genomic regions and entire genomes of viruses cloned on specific vectors. This study expands the use of Mu in vitro transposition in functional genetics and genomics by describing novel methods applicable to the targeted transgenesis of mouse and the whole-genome analysis of bacteriophages. The methods described here are rapid, efficient, and easily applicable to a wide variety of organisms, demonstrating the potential of the Mu transposition technology in the functional analysis of genes and genomes. First, an easy-to-use, rapid strategy to generate construct for the targeted mutagenesis of mouse genes was developed. To test the strategy, a gene encoding a neuronal K+/Cl- cotransporter was mutagenised. After a highly efficient transpositional mutagenesis, the gene fragments mutagenised were cloned into a vector backbone and transferred into bacterial cells. These constructs were screened with PCR using an effective 3D matrix system. In addition to traditional knock-out constructs, the method developed yields hypomorphic alleles that lead into reduced expression of the target gene in transgenic mice and have since been used in a follow-up study. Moreover, a scheme is devised to rapidly produce conditional alleles from the constructs produced. Next, an efficient strategy for the whole-genome analysis of bacteriophages was developed based on the transpositional mutagenesis of uncloned, infective virus genomes and their subsequent transfer into susceptible host cells. Mutant viruses able to produce viable progeny were collected and their transposon integration sites determined to map genomic regions nonessential to the viral life cycle. This method, applied here to three very different bacteriophages, PRD1, ΦYeO3 12, and PM2, does not require the target genome to be cloned and is directly applicable to all DNA and RNA viruses that have infective genomes. The method developed yielded valuable novel information on the three bacteriophages studied and whole-genome data can be complemented with concomitant studies on individual genes. Moreover, end-modified transposons constructed for this study can be used to manipulate genomes devoid of suitable restriction sites.

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.

Structuring time and questioning to achieve tactical awareness in games lessons

Background: A paradigm shift in educational policy to create problem solvers and critical thinkers produced the games concept approach (GCA) in Singapore's Revised Syllabus for Physical Education (1999). A pilot study (2001) conducted on 11 primary school student teachers (STs) using this approach identified time management and questioning as two of the major challenges faced by novice teachers. Purpose: To examine the GCA from three perspectives: structure—lesson form in terms of teacher-time and pupil-time; product—how STs used those time fractions; and process—the nature of their questioning (type, timing, and target). Participants and setting: Forty-nine STs from three different PETE cohorts (two-year diploma, four-year degree, two-year post-graduate diploma) volunteered to participate in the study conducted during the penultimate week of their final practicum in public primary and secondary schools. Intervention: Based on the findings of the pilot study, PETE increased the emphasis on GCA content specific knowledge and pedagogical procedures. To further support STs learning to actualise the GCA, authentic micro-teaching experiences that were closely monitored by faculty were provided in schools nearby. Research design: This is a descriptive study of time-management and questioning strategies implemented by STs on practicum. Each lesson was segmented into a number of sub-categories of teacher-time (organisation, demonstration and closure) and pupil-time (practice time and game time). Questions were categorised as knowledge, technical, tactical or affective. Data collection: Each ST was video-taped teaching a GCA lesson towards the end of their final practicum. The STs individually determined the timing of the data collection and the lesson to be observed. Data analysis: Each lesson was segmented into a number of sub-categories of both teacher- and pupil-time. Duration recording using Noldus software (Observer 4.0) segmented the time management of different lesson components. Questioning was coded in terms of type, timing and target. Separate MANOVAs were used to measure the difference between programmes and levels (primary and secondary) in relation to time-management procedures and questioning strategies. Findings: No differences emerged between the programmes or levels in their time-management or questioning strategies. Using the GCA, STs generated more pupil time (53%) than teacher time (47%). STs at the primary level provided more technical practice, and those in secondary schools more small-sided game play. Most questions (58%) were asked during play or practice but were substantially low-order involving knowledge or recall (76%) and only 6.7% were open-ended or divergent and capable of developing tactical awareness. Conclusions: Although STs are delivering more pupil time (practice and game) than teacher-time, the lesson structure requires further fine-tuning to extend the practice task beyond technical drills. Many questions are being asked to generate knowledge about games but lack sufficient quality to enhance critical thinking and tactical awareness, as the GCA intends.

Type III Secretion System of Phytopathogenic Bacterium Pseudomonas syringae:From Gene to Function

The type III secretion system (T3SS) is an essential requirement for the virulence of many Gram-negative bacteria which infect plants, animals and men. Pathogens use the T3SS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cells, where the effectors subvert host defenses. The best candidates for directing effector protein traffic are the bacterial type III-associated appendages, called needles or pili. In plant pathogenic bacteria, the best characterized example of a T3SS-associated appendage is the HrpA pilus of the plant pathogen Pseudomonas syringae pv. tomato DC3000. The components of the T3SS in plant pathogens are encoded by a cluster of hrp (hypersensitive reaction and pathogenicity) genes. Two major classes of T3SS-secreted proteins are: harpin proteins such as HrpZ which are exported into extracellular space, and avirulence (Avr) proteins such as AvrPto which are translocated directly to the plant cytoplasm. This study deals with the structural and functional characterization of the T3SS-associated HrpA pilus and the T3SS-secreted harpins. By insertional mutagenesis analysis of HrpA, we located the optimal epitope insertion site in the amino-terminus of HrpA, and revealed the potential application of the HrpA pilus as a carrier of antigenic determinants for vaccination. By pulse-expression of proteins combined with immuno-electron microscopy, we discovered the Hrp pilus assembly strategy as addition of HrpA subunits to the distal end of the growing pilus, and we showed for the first time that secretion of HrpZ occurs at the tip of the pilus. The pilus thus functions as a conduit delivering proteins to the extracellular milieu. By using phage-display and scanning-insertion mutagenesis methods we identified a conserved HrpZ-binding peptide and localized the peptide-binding site to the central domain of HrpZ. We also found that the HrpZ specifically interacts with a host bean protein. Taken together, the current results provide deeper insight into the molecular mechanism of T3SS-associated pilus assembly and effector protein translocation, which will be helpful for further studies on the pathogenic mechanisms of Gram-negative bacteria and for developing new strategies to prevent bacterial infection.

Self-assembly of hydrophobin proteins from the fungus Trichoderma reesei

Hydrophobins are small surface active proteins that are produced by filamentous fungi. The surface activity of hydrophobin proteins leads to the formation of a film at the air-water interface and adsorption to surfaces. The formation of these hydrophobin films and coatings is important in many stages of fungal development. Furthermore, these properties make hydrophobins interesting for potential use in technical applications. The surfactant-like properties of hydrophobins from Trichoderma reesei were studied at the air-water interface, at solid surfaces, and in solution. The hydrophobin HFBI was observed to spontaneously form a cohesive film on a water drop. The film was imaged using atomic force microscopy from both sides, revealing a monomolecular film with a defined molecular structure. The use of hydrophobins as surface immobilization carriers for enzymes was studied using fusion proteins of HFBI or HFBII and an enzyme. Furthermore, sitespecifically modified variants of HFBI were shown to retain their ability to selfassemble at interfaces and to be able to bind a second layer of proteins by biomolecular recognition. In order to understand the function of hydrophobins at interfaces, an understanding of their overall behavior and self-assembly is needed. HFBI and HFBII were shown to associate in solution into dimers and tetramers in a concentration-dependent manner. The association dynamics and protein-protein interactions of HFBI and HFBII were studied using Förster resonance energy transfer and size exclusion chromatography. It was shown that the surface activity of HFBI is not directly dependent on the formation of multimers in solution.

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.

Unravelling Molecular and Cellular Disease Mechanisms in Infantile Neuronal Ceroid Lipofuscinosis (INCL)

Studying neurodegeneration provides an opportunity to gain insights into normal cell physiology, and not just pathophysiology. In this thesis work the focus is on Infantile Neuronal Ceroid Lipofuscinosis (INCL). It is a recessively inherited lysosomal storage disorder. The disease belongs to the neuronal ceroid lipofuscinoses (NCLs), a group of common progressive neurodegenerative diseases of the childhood. Characteristic accumulation of autofluorescent storage material is seen in most tissues but only neurons of the central nervous system are damaged and eventually lost during the course of the disease leaving most other cell types unaffected. The disease is caused by mutations in the CLN1 gene, but the physiological function of the corresponding protein the palmitoyl protein thioesterase (PPT1) has remained elusive. The aim of this thesis work was to shed light on the molecular and cell biological mechanisms behind INCL. This study pinpointed the localization of PPT1 in axonal presynapses of neurons. It also established the role of PPT1 in early neuronal maturation as well as importance in mature neuronal synapses. This study revealed an endocytic defect in INCL patient cells manifesting itself as delayed trafficking of receptor and non-receptor mediated endocytic markers. Furthermore, this study was the first to connect the INCL storage proteins the sphingolipid activator proteins (SAPs) A and D to pathological events on the cellular level. Abnormal endocytic processing and intracellular re-localization was demonstrated in patient cells and disease model knock-out mouse neurons. To identify early affected cellular and metabolic pathways in INCL, knock-out mouse neurons were studied by global transcript profiling and functional analysis. The gene expression analysis revealed changes in neuronal maturation and cell communication strongly associated with the regulated secretory system. Furthermore, cholesterol metabolic pathways were found to be affected. Functional studies with the knock-out mouse model revealed abnormalities in neuronal maturation as well as key neuronal functions including abnormalities in intracellular calcium homeostasis and cholesterol metabolism. Together the findings, introduced in this thesis work, support the essential role of PPT1 in developing neurons as well as synaptic sites of mature neurons. Results of this thesis also elucidate early events in INCL pathogenesis revealing defective pathways ultimately leading to the neurodegenerative process. These results contribute to the understanding of the vital physiological function of PPT1 and broader knowledge of common cellular mechanisms behind neurodegeneration. These results add to the knowledge of these severe diseases offering basis for new approaches in treatment strategies.

Phospholipids of lipid-containing bacteriophages and their transbilayer distribution

In this study we used electro-spray ionization mass-spectrometry to determine phospholipid class and molecular species compositions in bacteriophages PM2, PRD1, Bam35 and phi6 as well as their hosts. To obtain compositional data of the individual leaflets, phospholipid transbilayer distribution in the viral membranes was studied. We found that 1) the membranes of all studied bacteriophage are enriched in PG as compared to the host membranes, 2) molecular species compositions in the phage and host membranes are similar, and 3) phospholipids in the viral membranes are distributed asymmetrically with phosphatidylglycerol enriched in the outer leaflet and phosphatidylethanolamine in the inner one (except Bam35). Alternative models for selective incorporation of phospholipids to phages and for the origins of the asymmetric phospholipid transbilayer distribution are discussed. Notably, the present data are also useful when constructing high resolution structural models of bacteriophages, since diffraction methods cannot provide a detailed structure of the membrane due to high motility of the lipids and lack of symmetric organization of membrane proteins.