Cellular Membranes as a Playground for Semliki Forest Virus Replication Complex

All positive-strand RNA viruses utilize cellular membranes for the assembly of their replication complexes, which results in extensive membrane modification in infected host cells. These alterations act as structural and functional scaffolds for RNA replication, providing protection for the viral double-stranded RNA against host defences. It is known that different positive-strand RNA viruses alter different cellular membranes. However, the origin of the targeted membranes, the mechanisms that direct replication proteins to specific membranes and the steps in the formation of the membrane bound replication complex are not completely understood. Alphaviruses (including Semliki Forest virus, SFV), members of family Togaviridae, replicate their RNA in association with membranes derived from the endosomal and lysosomal compartment, inducing membrane invaginations called spherules. Spherule structures have been shown to be the specific sites for RNA synthesis. Four replication proteins, nsP1-nsP4, are translated as a polyprotein (P1234) which is processed autocatalytically and gives rise to a membrane-bound replication complex. Membrane binding is mediated via nsP1 which possesses an amphipathic α-helix (binding peptide) in the central region of the protein. The aim of this thesis was to characterize the association of the SFV replication complex with cellular membranes and the modification of the membranes during virus infection. Therefore, it was necessary to set up the system for determining which viral components are needed for inducing the spherules. In addition, the targeting of the replication complex, the formation site of the spherules and their intracellular trafficking were studied in detail. The results of current work demonstrate that mutations in the binding peptide region of nsP1 are lethal for virus replication and change the localization of the polyprotein precursor P123. The replication complex is first targeted to the plasma membrane where membrane invaginations, spherules, are induced. Using a specific regulated endocytosis event the spherules are internalized from the plasma membrane in neutral carrier vesicles and transported via an actin-and microtubule-dependent manner to the pericentriolar area. Homotypic fusions and fusions with pre-existing acidic organelles lead to the maturation of previously described cytopathic vacuoles with hundreds of spherules on their limiting membranes. This work provides new insights into the membrane binding mechanism of SFV replication complex and its role in the virus life cycle. Development of plasmid-driven system for studying the formation of the replication complex described in this thesis allows various applications to address different steps in SFV life cycle and virus-host interactions in the future. This trans-replication system could be applied for many different viruses. In addition, the current work brings up new aspects of membranes and cellular components involved in SFV replication leading to further understanding in the formation and dynamics of the membrane-associated replication complex.

Molecular mechanisms of bacteriophage phi6 RNA-dependent RNA polymerase and its utilization in biotechnology

Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.

Kognitiva system i namngivningen av finländska handelsfartyg 1838-1938

This dissertation is an onomastic study of Finland s stock of ship names (nautonomasticon) recorded over the period 1838 1938. The primary material investigated consists of 2 066 examples of ship names from the fleets of coastal towns, distributed over five sample years. The material is supplemented with two bodies of comparative data; one that consists of 2 535 examples of boat names from the archipelago area at the corresponding time, and another that comprises 482 examples of eighteenth century Finnish ship names. This study clarifies the categories of names that appear the frequency of the names, formation, morphology, linguistic origin, functions, and semantic qualities. By comparing the material with boat names from previous centuries, and from other countries, the characteristics of Finnish vessel names are further highlighted. Additional clarification is brought to the chronological, regional, and social variations, and to the emergence of various forms of systematic naming. This dissertation builds on older research from other countries, and uses traditional onomastic methods alongside a more modern methodology. The approach is interdisciplinary, meaning that the names are explored using facts not only from nautical history, but also from a range of other historical disciplines such as economics, culture, art, and literature. In addition, the approach is socio-onomastic, i.e. that the variations in names are studied in a societal context. Using a synchronised perspective, cognitive linguistic theories have provided the tools for this exploration into the metaphorical and the prototypical meaning of the names, and the semantic domains that the names create. The quantitative analysis has revealed the overall picture of Finnish boat names. Personal names, names from mythology, and place names, emerge as significant categories, alongside nonproprial names in Swedish and Finnish. The interdisciplinary perspective has made it possible to explain certain trends in the stock of boat names, for example, the predisposition towards names from classical mythology, the breakthrough of names taken from the national epos Kalevala, names in the Finnish language from around the middle of the nineteenth century, and the continuing rise of place names during the latter part of the period 1838 1938. The socio-onomastic perspective has also identified clear differences between those ship names used in towns, and those ship names used in the archipelago, and it has clarified how naming conventions tend to spread from town centres to peripheral areas. The cognitive linguistic methods have revealed that the greater part of the vessel names can be interpreted as metaphors, in particular personifications, and that many names are related in their content and also form semantic networks and cognitive systems. The results indicate that there is a mental nautonomasticon that consists of a standard set of traditional ship names, but they also reveal the existence of conscious or unconscious cognitive systems (rules and conventions) that guide the naming of boats.

Processing of Stimulus Repetition and Change in the Somatosensory System: Recordings of Electrical and Magnetic Brain Responses

In the present work, effects of stimulus repetition and change in a continuous stimulus stream on the processing of somatosensory information in the human brain were studied. Human scalp-recorded somatosensory event-related potentials (ERPs) and magnetoencephalographic (MEG) responses rapidly diminished with stimulus repetition when mechanical or electric stimuli were applied to fingers. On the contrary, when the ERPs and multi-unit a ctivity (MUA) were directly recorded from the primary (SI) and secondary (SII) somatosensory cortices in a monkey, there was no marked decrement in the somatosensory responses as a function of stimulus repetition. These results suggest that this rate effect is not due to the response diminution in the SI and SII cortices. Obviously the responses to the first stimulus after a long "silent" period are nhanced due to unspecific initial orientation, originating in more broadly distributed and/or deeper neural structures, perhaps in the prefrontal cortices. With fast repetition rates not only the late unspecific but also some early specific somatosensory ERPs were diminished in amplitude. The fast decrease of the ERPs as a function of stimulus repetition is mainly due to the disappearance of the orientation effect and with faster repetition rates additively due to stimulus specific refractoriness. A sudden infrequent change in the continuous stimulus stream also enhanced somatosensory MEG responses to electric stimuli applied to different fingers. These responses were quite similar to those elicited by the deviant stimuli alone when the frequent standard stimuli were omitted. This enhancement was obviously due to the release from refractoriness because the neural structures generating the responses to the infrequent deviants had more time to recover from the refractoriness than the respective structures for the standards. Infrequent deviant mechanical stimuli among frequent standard stimuli also enhanced somatosensory ERPs and, in addition, they elicited a new negative wave which did not occur in the deviants-alone condition. This extra negativity could be recorded to deviations in the stimulation site and in the frequency of the vibratory stimuli. This response is probably a somatosensory analogue of the auditory mismatch negativity (MMN) which has been suggested to reflect a neural mismatch process between the sensory input and the sensory memory trace.

Mutations of mitochondrial DNA polymerase gamma: an important cause of neurological disorders

Thesis focuses on mutations of POLG1 gene encoding catalytic subunit polγ-α of mitochondrial DNA polymerase gamma holoenzyme (polG) and the association of mutations with different clinical phenotypes. In addition, particular defective mutant variants of the protein were characterized biochemically in vitro. PolG-holoenzyme is the sole DNA polymerase found in mitochondria. It is involved in replication and repair of the mitochondrial genome, mtDNA. Holoenzyme also includes the accessory subunit polγ-β, which is required for the enhanced processivity of polγ-α. Defective polγ-α causes accumulation of secondary mutations on mtDNA, which leads to a defective oxidative phosphorylation system. The clinical consequences of such mutations are variable, affecting nervous system, skeletal muscles, liver and other post-mitotic tissues. The aims of the studies included: 1) Determination of the role of POLG1 mutations in neurological syndromes with features of mitochondrial dysfunction and an unknown molecular cause. 2) Development and set up of diagnostic tests for routine clinical purposes. 3) Biochemical characterization of the functional consequences of the identified polγ-α variants. Studies describe new neurological phenotypes in addition to PEO caused by POLG1 mutations, including parkinsonism, premature amenorrhea, ataxia and Parkinson s disease (PD). POLG1 mutations and polymorphisms are both common and/or potential genetic risk factors at least among the Finnish population. The major findings and applications reported here are: 1) POLG1 mutations cause parkinsonism and premature menopause in PEO families in either a recessive or a dominant manner. 2) A common recessive POLG1 mutations (A467T and W748S) in the homozygous state causes severe adult or juvenile-onset ataxia without muscular symptoms or histological or mtDNA abnormalities in muscles. 3) A common recessive pathogenic change A467T can also cause a mild dominant disease in heterozygote carriers. 4) The A467T variant shows reduced polymerase activity due to defective template binding. 5) Rare polyglutamine tract length variants of POLG1 are significantly enriched in Finnish idiopathic Parkinson s disease patients. 6) Dominant mutations are clearly restricted to the highly conserved polymerase domain motifs, whereas recessive ones are more evenly distributed along the protein. The present results highlight and confirm the new role of mitochondria in parkinsonism/Parkinson s disease and describe a new mitochondrial ataxia. Based on these results, a POLG1 diagnostic routine has been set up in Helsinki University Central Hospital (HUSLAB).

Exact bounds for distributed graph colouring

A distributed system is a collection of networked autonomous processing units which must work in a cooperative manner. Currently, large-scale distributed systems, such as various telecommunication and computer networks, are abundant and used in a multitude of tasks. The field of distributed computing studies what can be computed efficiently in such systems. Distributed systems are usually modelled as graphs where nodes represent the processors and edges denote communication links between processors. This thesis concentrates on the computational complexity of the distributed graph colouring problem. The objective of the graph colouring problem is to assign a colour to each node in such a way that no two nodes connected by an edge share the same colour. In particular, it is often desirable to use only a small number of colours. This task is a fundamental symmetry-breaking primitive in various distributed algorithms. A graph that has been coloured in this manner using at most k different colours is said to be k-coloured. This work examines the synchronous message-passing model of distributed computation: every node runs the same algorithm, and the system operates in discrete synchronous communication rounds. During each round, a node can communicate with its neighbours and perform local computation. In this model, the time complexity of a problem is the number of synchronous communication rounds required to solve the problem. It is known that 3-colouring any k-coloured directed cycle requires at least ½(log* k - 3) communication rounds and is possible in ½(log* k + 7) communication rounds for all k ≥ 3. This work shows that for any k ≥ 3, colouring a k-coloured directed cycle with at most three colours is possible in ½(log* k + 3) rounds. In contrast, it is also shown that for some values of k, colouring a directed cycle with at most three colours requires at least ½(log* k + 1) communication rounds. Furthermore, in the case of directed rooted trees, reducing a k-colouring into a 3-colouring requires at least log* k + 1 rounds for some k and possible in log* k + 3 rounds for all k ≥ 3. The new positive and negative results are derived using computational methods, as the existence of distributed colouring algorithms corresponds to the colourability of so-called neighbourhood graphs. The colourability of these graphs is analysed using Boolean satisfiability (SAT) solvers. Finally, this thesis shows that similar methods are applicable in capturing the existence of distributed algorithms for other graph problems, such as the maximal matching problem.